Method for preparing macrocyclodextrin from high-concentration substrate

Abstract

The invention discloses a method for preparing macrocyclodextrin from a high-concentration substrate, and belongs to the technical field of starch processing. The method comprises the following steps: gelatinizing and liquefying a high-concentration starch suspension (30%, w / w) by using 4-alpha-glycosyltransferase as a liquefying enzyme, carrying out enzyme deactivation treatment after liquefying, simultaneously carrying out debranching and cyclization reaction on the starch by using pullulanase and 4-alpha-glycosyltransferase, carrying out enzyme deactivation and centrifugal treatment after the reaction is completed, and carrying out freeze-drying to obtain the starch. And carrying out beta-amylase purification treatment, alcohol precipitation, centrifugation and dialysis on the supernatant, carrying out freeze drying, and collecting the target product LR-CDs with the yield of 23.78%. According to the method, the high-concentration starch is used as the substrate for preparing the LR-CDs for the first time, experiments prove that the preparation efficiency of preparing the LR-CDs from the starch can be greatly improved through the preparation technology, the product purity is high, and the method has the potential of achieving industrial large-scale production of the LR-CDs.

Description

Technical Field

[0001] The invention relates to a method for preparing macrocyclodextrin from a high-concentration substrate, belonging to the technical field of food industry. Background Art

[0002] Large ring cyclodextrins (LR-CDs) are cyclic α-1,4-glucans composed of 9 to hundreds of pyranose units. LR-CDs are mainly obtained by intramolecular transglycosylation of amylose through the cyclization of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) or 4-α-glucanotransferase (4αGT, EC 2.4.1.25). Compared with linear oligosaccharides, LR-CDs are hydrophilic on the outside and hydrophobic on the inside, and have higher water solubility. The water solubility of LR-CDs is higher than that of common α-CD, β-CD and γ-CD, which may be due to the high structural flexibility formed by intramolecular and intermolecular hydrogen bonds. At the same time, LR-CDs have relatively large hydrophobic cavities. Therefore, the large hydrophobic cavities can form inclusion complexes with some larger guest molecules. These characteristics make LR-CDs have great application potential in the fields of medicine, food, biology, etc.

[0003] Although some progress has been made in the enzymatic preparation of LR-CDs and the application of some of its properties, the bottleneck problem of low preparation yield still exists. It is only in the laboratory preparation stage, and it is still difficult to realize industrial preparation and application. The substrate is an important factor affecting the yield of LR-CD. The most suitable substrate for 4αGT is amylose. Generally, artificially synthesized potato amylose is used, but it is expensive, so the production cost is very high. If natural starch containing a large amount of amylopectin is directly used to prepare and produce LR-CDs, the yield is very low. At present, there is no preparation method that can meet the requirements of industrial mass production of LR-CDs.

[0004] In view of this, it is of great significance to find a method for preparing LR-CDs with the potential for industrial large-scale production. The present invention lays a good foundation for realizing large-scale industrial production of LR-CDs. Summary of the invention

[0005] In view of the high cost and low yield of LR-CDs preparation, which makes it difficult to achieve industrial mass production, the inventors have provided a method for preparing LR-CDs using high-concentration substrates through research and improvement. This method has the potential for industrial large-scale production. Compared with common α, β, and γ-cyclodextrins, LR-CDs have larger cavity sizes and exhibit unique structures and physical and chemical properties. These properties make LR-CDs show great application potential in the fields of food, medicine, biology, etc.

[0006] The invention provides a method for preparing LR-CDs by using a high-concentration substrate. The method comprises the following steps: firstly liquefying starch with a liquefaction enzyme, inactivating the enzyme after liquefaction is completed, and then simultaneously performing debranching and cyclization reactions on the starch with a debranching enzyme and a glycosyltransferase, inactivating the enzyme after the reaction is completed, centrifuging the starch, purifying the supernatant with amylase, and performing alcohol precipitation, centrifugation, and dialysis before drying to obtain the target product LR-CDs.

[0007] The present invention also provides a method for preparing LR-CDs, the method comprising the following steps:

[0008] (1) adding a liquefaction enzyme to a starch suspension to simultaneously carry out a liquefaction reaction during the gelatinization process; the mass fraction (w / w) of starch in the starch suspension is greater than 7%, preferably greater than 10%, and more preferably greater than 20%;

[0009] (2) adding a debranching enzyme and a cyclase to the starch obtained in step (1) at the same time, and reacting them to obtain a reaction product; or first adding a debranching enzyme to the starch obtained in step (1) to carry out a debranching reaction, and then adding a cyclase to carry out a reaction to obtain a reaction product;

[0010] (3) Adding amylase to the reaction product obtained in step (2) to carry out reaction to prepare LR-CDs.

[0011] In one embodiment of the present invention, the mass fraction (w / w) of starch in the starch suspension is 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, 19% to 20%, 21% to 22%, 22% to 23%, 23% to 24%, 24% to 25%, 25% to 26%, 26% to 27%, 27% to 28%, 28% to 29%, 30% to 31%, 31% to 32%, 32% to 33%, 33% to 34%, 35% to 36%, 37% to 38%, 39% to 40%, 40% to 41%, 41% to 42%, 42% to 43%, 44% to 45%, 45% to 46%, 47% to 48%, 49% to 50%, 50% to 51%, 51% to 52%, 52% to 53%, 53% to 54%, 54% to 55%, 56% to 57%, 58% to 59%, 60% to 61%, 61% to 62%, 62% to 63%, 64% to 65%, 66% to 67%, 68% to 69%, 70% to 71%, 71% to 72%, 72% to 73%, 73% to 74%, 75% to 76%, 77% to 78%, 79% to 8 7%, 27%-28%, 28%-29%, 29%-30%, 31%-32%, 32%-33%, 33%-34%, 34%-35%, 35%-36%, 36%-37%, 37%-38%, 38%-39%, 39%-40%, 40%-41%, 41%-42%, 42%-43%, 43%-44%, 44%-45%, 45%-46%, 46%-47%, 47%-48%, 48%-49% or 49%-50%.

[0012] In one embodiment of the present invention, the types of starch substrates include potato starch, tapioca starch, corn starch, pea starch and the like.

[0013] In one embodiment of the present invention, the liquefaction enzyme includes but is not limited to: α-amylase, 4-α-glycosyltransferase, cyclodextrin glucosyltransferase.

[0014] In one embodiment of the present invention, the liquefaction enzyme includes but is not limited to: α-amylase (EC 3.2.1.1), 4-α-glycosyltransferase (EC 2.4.1.25), cyclodextrin glucosyltransferase (EC 2.4.1.19).

[0015] In one embodiment of the present invention, when the liquefying enzyme is 4-α-glycosyltransferase, the added amount is: calculated on the basis of substrate, 1U / g starch to 20U / g starch;

[0016] In one embodiment of the present invention, when the liquefying enzyme is α-amylase, the added amount is: calculated on the basis of substrate, 1U / g starch to 20U / g starch;

[0017] In one embodiment of the present invention, when the liquefying enzyme is cyclodextrin glucosyltransferase, the added amount is: calculated on the basis of substrate, 1U / g starch to 20U / g starch.

[0018] In one embodiment of the present invention, in step (1), the liquefaction reaction is carried out simultaneously with the gelatinization process by adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, heating the mixture to 80-100° C. and maintaining the temperature for 20-60 min;

[0019] Preferably, after the liquefaction reaction is carried out simultaneously during the gelatinization process, the DE value of the starch is controlled to be 2-8.

[0020] In one embodiment of the present invention, the addition amount of the liquefaction enzyme is: 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 15U / g starch, 16U / g starch to 17U / g starch, 18U / g starch to 20U / g starch, 19U / g starch to 21U / g starch, 22U / g starch to 23U / g starch, 24U / g starch to 25U / g starch, 26U / g starch to 27U / g starch, 28U / g starch to 29U / g starch, 30U / g starch to 31U / g starch, 32U / g starch to 33U / g starch, 34U / g starch to 35U / g starch, 36U / g starch to 37U / g starch, 38U / g starch to 39U / g starch, 40U / g starch to 41U / g starch, 42U / g starch to 43U / g starch, 44U / g starch to 45U / g starch, 46U / g starch to 47U / g starch, g starch ~ 11U / g starch, 11U / g starch ~ 12U / g starch, 12U / g starch ~ 13U / g starch, 13U / g starch ~ 14U / g starch, 14U / g starch ~ 15U / g starch, 15U / g starch ~ 16U / g starch, 16U / g starch ~ 17U / g starch, 17U / g starch ~ 18U / g starch, 18U / g starch ~ 19U / g starch or 19U / g starch ~ 20U / g starch.

[0021] In one embodiment of the present invention, the liquefaction enzyme is a 4-α-glycosyltransferase, and the 4-α-glycosyltransferase can be derived from: Deinococcus geothermalis, Aerococcus, Thermus filamentous, Escherichia coli, Thermusancient, Thermus lettrichia, Thermus aquaticus, Streptococcus agalactiae, Corynebacterium glutamicum, Synechococcus PCC 6803, Synechococcus PCC 6804, Manihotesculenta, Arabidopsis thaliana, and Solanum tuberosum.

[0022] In one embodiment of the present invention, in step (1), the liquefaction reaction is carried out simultaneously during the gelatinization process, and after adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, the temperature is raised to 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or 90°C, and maintained for 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min or 60min.

[0023] In one embodiment of the present invention, the debranching enzyme includes but is not limited to: pullulanase, isoamylase, pullulanase; the glycosyltransferase includes but is not limited to: 4-α-glycosyltransferase, cyclodextrin glucosyltransferase.

[0024] In one embodiment of the present invention, the debranching enzyme includes but is not limited to: pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.57).

[0025] In one embodiment of the present invention, the amount of the debranching enzyme added is, based on the substrate, at least 100 U / g of starch; the amount of the cyclase added is: based on the substrate, 1 U / g starch to 20 U / g starch.

[0026] In one embodiment of the present invention, the debranching conditions are: adding 10 to 1000 U / g of starch debranching enzyme, and performing enzymolysis at 40 to 80° C. for 1 to 24 hours;

[0027] In one embodiment of the present invention, the cyclase includes but is not limited to: 4-α-glycosyltransferase (EC 2.4.1.25) and cyclodextrin glucosyltransferase (EC 2.4.1.19).

[0028] In one embodiment of the present invention, the cyclization conditions are: adding 1 to 100 U / g starch cyclase, and enzymolysis at 40 to 80° C. for 1 to 24 hours;

[0029] In one embodiment of the present invention, the addition amount of the cyclase is: 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 11U / g starch, 11U / g starch to 12U / g starch, 12U / g starch to 13U / g starch, 13U / g starch to 14U / g starch powder, 14U / g starch ~ 15U / g starch, 15U / g starch ~ 16U / g starch, 16U / g starch ~ 17U / g starch, 17U / g starch ~ 18U / g starch, 18U / g starch ~ 19U / g starch, 19U / g starch ~ 20U / g starch, 20U / g starch ~ 30U / g starch, 30U / g starch ~ 40U / g, 40U / g starch ~ 50U / g, 50U / g starch ~ 60U / g, 60U / g starch ~ 70U / g, 70U / g starch ~ 80U / g, 80U / g starch ~ 90U / g or 90U / g starch ~ 100U / g.

[0030] In one embodiment of the present invention, in step (2), the reaction conditions are: reaction at 50°C to 80°C for 1 to 48 hours;

[0031] In one embodiment of the present invention, the temperature is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C or 80°C;

[0032] In one embodiment of the invention, the reaction time is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h or 48 h.

[0033] In one embodiment of the present invention, in step (3), the amylase is β-amylase or saccharifying enzyme; the amount of the amylase added is, calculated on the basis of the substrate, 1U / g starch to 100U / g starch; the reaction temperature after adding the amylase is 30°C to 60°C, and the reaction time is 1 to 48h.

[0034] In one embodiment of the present invention, the amylase includes but is not limited to: β-amylase (EC 3.2.1.2), saccharifying enzyme (EC 3.2.1.3).

[0035] In one embodiment of the present invention, the conditions for the amylase treatment are: adding 1 to 100 U / g of starch amylase, and enzymolysis at 40 to 80° C. for 1 to 24 hours;

[0036] In one embodiment of the present invention, the temperature is 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C.

[0037] In one embodiment of the present invention, the time is 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h or 48h.

[0038] In one embodiment of the present invention, step (3) also includes a purification and separation step, and the purification and separation method is to obtain the product after alcohol precipitation and dialysis drying.

[0039] In one embodiment of the present invention, the purification step is to prepare LR-CDs after amylase treatment by centrifugation, rotary evaporation, alcohol precipitation, centrifugation, and dialysis;

[0040] In one embodiment of the present invention, the enzyme inactivation method includes high temperature enzyme inactivation, acid-base enzyme inactivation, and ethanol enzyme inactivation.

[0041] In one embodiment of the present invention, ethanol is used for the alcohol precipitation, and the ratio of the reaction solution to the ethanol is 1:1 to 1:10.

[0042] In one embodiment of the present invention, the molecular weight cut-off of the dialysis bag is 500 to 3000 Da.

[0043] In one embodiment of the present invention, the drying method includes freeze drying, atmospheric pressure drying, spray drying, drum drying or microwave drying.

[0044] The present invention provides LR-CDs prepared by the above method.

[0045] The present invention also provides applications of the LR-CDs in the fields of food, medicine and biology.

[0046] In one embodiment of the present invention, the application of LR-CDs in the field of food includes improving the flavor and texture of food and inhibiting food retrogradation. During the food processing, some foods themselves have unpleasant flavors such as bitterness and fishy smell. The hydrophobic cavities of LR-CDs can be used to embed the mixture with foreign smell and remove the foreign taste in the food; at the same time, because LR-CDs have the characteristic of not being easy to retrograde, they can be used as retrogradation inhibitors for ready-to-eat foods such as bread, steamed buns and dumplings; in addition, LR-CDs can delay the oxidation and enzymatic degradation of phenolic compounds, and LR-CDs can be added to fruit juice as an anti-browning agent to prevent the oxidation and discoloration of the fruit juice.

[0047] In one embodiment of the present invention, the application of the LR-CDs in the medical field includes improving the physicochemical properties of drugs, improving stability, solubility and bioavailability. Compared with other polymer carriers, LR-CDs have strong advantages because they are natural materials, generally have good biocompatibility, biodegradability and are safe and non-toxic.

[0048] In one embodiment of the present invention, the LR-CDs are used in the biological field as an artificial chaperone for protein chaperones and added to commercial protein refolding kits to prevent protein aggregation and improve its folding degree, and can promote the denatured enzyme protein to refold correctly and restore the activity of the enzyme protein.

[0049] The present invention provides the use of the method or the LR-CDs prepared by the method in food, biological products and pharmaceuticals.

[0050] Beneficial Effects

[0051] The present invention is the first to prepare LR-CDs using high-concentration starch as a substrate (the concentration can reach 30% w / w). Experiments have shown that 4-α-glycosyltransferase can be used to liquefy high-concentration starch, which makes it possible to prepare LR-CDs using high-concentration substrates. Through the preparation process of the present invention, the yield of LR-CDs prepared from starch is greatly improved, and the product purity is high, which has the potential to realize industrial large-scale production of LR-CDs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : LC-MS identification chart of LR-CDs prepared by synchronous reaction of pullulanase and 4-α-glycosyltransferase for 12 h.

[0053] Figure 2 : LC-MS identification chart of LR-CDs prepared by synchronous reaction of pullulanase and 4-α-glycosyltransferase for 24 hours.

[0054] Figure 3 :Effect of the amount of 4-α-glycosyltransferase added in liquefaction reaction on the yield of LR-CDs.

[0055] Figure 4 : Effect of pullulanase dosage on LR-CDs yield.

[0056] Figure 5 :Effect of the amount of 4-α-glycosyltransferase added in the cyclization reaction on the yield of LR-CDs.

[0057] Figure 6 :Effect of β-amylase dosage on LR-CDs yield. DETAILED DESCRIPTION

[0058] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0059] As used herein, the term "about" indicates that a value includes the inherent variation of error for the method being employed to determine the value, or variation that exists between experiments.

[0060] Methods for preparing LR-CDs are described in this disclosure.

[0061] The calculation method of the yield involved in the following examples is:

[0062] Yield = mass of final product / mass of initial starch substrate × 100%.

[0063] Technical terms:

[0064] Large ring cyclodextrins (LR-CDs) are cyclic α-1,4-glucans composed of 9 to hundreds of pyranose units. LR-CDs are mainly obtained by intramolecular transglycosylation of amylose through the cyclization of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) or 4-α-glucanotransferase (4αGT, EC 2.4.1.25). Compared with common α, β, and γ-cyclodextrins, LR-CDs have larger cavity sizes and exhibit unique structural and physicochemical properties. These properties make LR-CDs show great application potential in the fields of food, medicine, and biology.

[0065] The method for preparing LR-CDs of the present invention comprises the following steps: adding 4-α-glycosyltransferase to carry out liquefaction reaction during starch gelatinization, adding debranching enzyme and cyclase to carry out synchronous action after the reaction is completed, adding amylase to the obtained reaction product to carry out purification reaction, and obtaining LR-CDs after a purification step.

[0066] Or after gelatinizing the starch, add 4-α-glycosyltransferase to carry out liquefaction reaction. After the reaction is completed, add debranching enzyme to the obtained starch for debranching, and then add glycosyltransferase to react. Add amylase to the obtained reaction product for purification reaction, and obtain LR-CDs after a purification step.

[0067] On the one hand, when preparing large cyclodextrins (LR-CDs), all starches are suitable for the present invention as substrates. Starch can be derived from any natural source, and "natural" starch is an unmodified form found in nature. Starch is a carbohydrate widely found in plants, mainly synthesized by glucose molecules through starch bodies and stored in the roots, stems, leaves and seeds of plants. Depending on the different plant sources, the types and properties of starch are also different. Natural sources can be cereal starch, bean starch, potato starch, etc., such as corn, peas, potatoes, sweet potatoes, barley, wheat, rice, cassava, sorghum, lotus root, yam, etc.

[0068] In one aspect, as used herein, the term "pea starch" refers to starch prepared from pea (Pisum Sativum) seeds. In one aspect, pea starch is prepared from garden pea (Pisum Sativum ssp. Hortense). In another aspect, pea starch is prepared from red pea (Pisum Sativum ssp. arvense). In one aspect, pea starch is prepared from fresh peas. In another aspect, pea starch is prepared from frozen peas. In one aspect, pea starch does not include a significant amount of protein. In one aspect, pea starch includes no more than 5% (w / w) protein. In one aspect, pea starch does not include a significant amount of fiber. In one aspect, pea starch includes an amylose content greater than 25% (w / w). In another aspect, pea starch includes an amylose content greater than 30% (w / w). In another aspect, pea starch includes an amylose content greater than 35% (w / w).

[0069] In one aspect, the starch can be reacted in the form of a starch suspension, wherein the mass fraction of starch in the starch suspension is greater than 7%, preferably greater than 10%.

[0070] On the one hand, the starch can be reacted in the form of a starch suspension, and the mass fraction of starch in the starch suspension can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%.

[0071] On the one hand, the starch can be reacted in the form of a starch suspension, and the mass fraction of starch in the starch suspension can be 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, 19% to 20%, 21% to 22%, 22% to 23%, 23% to 24%, 24% to 25%, 25% to 26%, 44%-45%, 45%-46%, 46%-47%, 47%-48%, 48%-49% or 49%-50%.

[0072] On the one hand, the gelatinization refers to the process in which starch particles are transformed from a solid state to a gel or paste state under the action of heating 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 an embodiment of the present invention, the gelatinization is to react under certain temperature conditions after adding water; in the present invention, the starch suspension can be gelatinized, and the temperature can be directly increased for gelatinization; different types of starch have different gelatinization temperatures, for example, the gelatinization temperature of corn starch is about 60°C to 80°C; the gelatinization temperature of potato starch is about 60°C to 80°C; the gelatinization temperature of wheat starch is about 60°C to 70°C; the gelatinization temperature of rice starch is about 70°C to 85°C; the gelatinization temperature of lotus seed starch is about 70°C to 80°C.

[0073] In laboratory environments, a water bath is often used. However, in actual industrial production and food processing, the technical solution of the present invention can be achieved as long as water is added to starch and the temperature is raised to perform gelatinization reaction.

[0074] In one aspect, the present invention adopts the method of first subjecting the gelatinized starch to a liquefaction reaction;

[0075] Starch liquefaction reaction:

[0076] Refers to the process of breaking starch molecular chains into shorter fragments so that they can be dissolved in water or other solvents.

[0077] The starch liquefaction method and principle that can be used in the present invention is enzymatic liquefaction: using amylase (such as α-amylase, starch phosphatase, etc.) to break the starch molecular chain into small fragments. These enzymes can react with starch under appropriate temperature and pH conditions to break the α-1,4-glycosidic bonds in the starch molecular chain and form dextrin, dextrin fragments and oligosaccharides.

[0078] The liquefied starch can be used in food, feed, textile, medicine and other industries to improve the utilization rate and functionality of starch.

[0079] In one aspect, the present invention uses 4-α-glycosyltransferase to carry out the liquefaction reaction.

[0080] 4-α-Glycosyltransferase

[0081] 4αGT (EC 2.4.1.25) belongs to the GH13, 57 and 77 families. Its sources are mainly divided into two categories: one is plant-derived, mainly from plant tubers, such as potato tubers. Plant-derived 4αGT is also known as D enzyme or dismutase. The main function of D enzyme is chain extension, which may be involved in starch metabolism and chain length modification (TAKAHA T, YANASE M, OKADA S, et al. Disproportionating enzyme (4-α-glucanotransferase; EC 2.4.1.25) of potato. Purification, molecular cloning, and potential role in starch metabolism[J]. Journal of Biological Chemistry, 1993, 268(2): 1391-1396. DOI: 10.1016 / S0021-9258(18)54088-6.). It can catalyze intramolecular transglycosylation to synthesize cyclodextrins with DP greater than 17, namely LR-CDs (SRISIMARAT W, POWVIRIYAKUL A, KAULPIBOON J, et al. A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrinproducts[J]. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 2010, 70(3-4): 369-375. DOI: 10.1007 / s10847-010-9890-5). The other type is from microorganisms, mainly from bacteria or archaea, and is an intracellular enzyme, also known as maltogenic amylase, which is related to maltose metabolism and glycogen synthesis or degradation in microorganisms (MACASKIE LE, PALMER TN. The role of amylomaltase and maltodextrinphosphorylase in the pathway of glycogen catabolism in Escherichia coli [proceedings] [J]. Biochemical Society transactions, 1978, 6 (2): 405-407. DOI: 10.1042 / bst0060405).Different from CGTases, the spatial structure of 4αGT is divided into A, B1, B2 and B3 domains. Domain A is also a barrel-shaped structure composed of 8 α-helices and 8 β-folds, and is the catalytic center of 4αGT; the 250s loop in domain B1 is unique to 4αGT (JUNG JH, JUNG TY, SEO DH, et al. Structural and functional analysis of substrate recognition by the 250s loop in amylomaltase from Thermus brockianus [J]. Proteins, 2011, 79 (2): 633-644. DOI: 10.1002 / prot.22911). Studies have shown that the steric hindrance of the 250s loop can limit the production of cyclic products with smaller DP (PRZYLAS I, TOMOO K, TERADA Y, et al. Crystal structure of amylomaltase from thermus aquaticus, aglycosyltransferase catalysing the production of large cyclic glucans [J]. Journal of molecular biology, 2000, 296(3):873-886. DOI:10.1006 / jmbi.1999.3503.), thereby controlling the minimum degree of polymerization of LR-CDs produced by 4αGT; domain B2 is believed to be related to the substrate specificity of the enzyme or the DP size of the product (Xu Yan. Research on the action of 4-α-glycosyltransferase on the preparation of LR-CDs by debranched starch [D]; Jiangnan University, 2014.).

[0082] Method for determining the activity of 4-α-glycosyltransferase: (1) Preparation of amylose standard solution: Accurately weigh 80 mg of potato amylose, add 20 mL of NaOH solution (1 mol / L), and mix well on a vortex oscillator for later use. When in use, take out an appropriate amount of amylose solution, add an equal volume of hydrochloric acid solution (1 mol / L) to neutralize to neutrality, and obtain a 0.2% (w / v) amylose standard solution. (2) Determination of amylose-iodine standard curve: Prepare amylose solutions of different concentrations. When determining, take 0.1 mL of each of the above solutions, add 1 mL of iodine solution (0.2% KI + 0.02% I2), mix well, let stand for 1 min at room temperature (25°C), and measure the absorbance at 620 nm. Draw a standard curve based on the relationship between the absorbance and the amylose concentration. (3) Take 300 μL of substrate (0.2% potato amylose solution + 1.0% maltose solution) and 600 μL of Tris-HCl buffer (20 mmol / L, pH 7.5) in a test tube, preheat at 75°C for 10 min, add 100 μL of enzyme solution (0.2 mg / mL), and react for 10 min. Inactivate the enzyme in a boiling water bath for 10 min to terminate the reaction, and cool in ice water. Take 0.1 mL of the reaction solution, add 1 mL of iodine solution and mix well, let stand at room temperature (25°C) for 1 min, measure its absorbance at 620 nm, and use no enzyme solution as a control. Under the above reaction conditions, the amount of enzyme required to degrade 0.5 mg / mL amylose per minute is defined as one enzyme activity unit (U).

[0083] On the one hand, the 4-α-glycosyltransferase can be derived from: Deinococcus geothermalis, Aerococcus, Thermus filamentous, Escherichia coli, Thermus ancient, Thermus lettrichia, Thermus aquaticus, Streptococcus agalactiae, Corynebacterium glutamicum, Synechococcus PCC 6803, Synechococcus PCC 6804, Manihot esculenta, Arabidopsis thaliana, Solanum tuberosum.

[0084] On the one hand, in the liquefaction reaction, the added amount of 4-α-glycosyltransferase, calculated based on the substrate, can be: 1 U / g starch, 2 U / g starch, 5 U / g starch, 10 U / g starch, 20 U / g starch.

[0085] On the one hand, in the liquefaction reaction, the added amount of 4-α-glycosyltransferase can be 1U / g starch, 2U / g starch, 3U / g starch, 4U / g starch, 5U / g starch, 6U / g starch, 7U / g starch, 8U / g starch, 9U / g starch, 10U / g starch, 11U / g starch, 12U / g starch, 13U / g starch, 14U / g starch, 15U / g starch, 16U / g starch, 17U / g starch, 18U / g starch, 19U / g starch or 20U / g starch.

[0086] In one aspect, in the liquefaction reaction, the amount of 4-α-glycosyltransferase added can be 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 11U / g starch, 12U / g starch to 13U / g starch, 14U / g starch to 15U / g starch, 16U / g starch to 17U / g starch, 18U / g starch to 19U / g starch, 20U / g starch to 21U / g starch, 22U / g starch to 23U / g starch, 24U / g starch to 25U / g starch, 26U / g starch to 27U / g starch, 28U / g starch to 29U / g starch, 30U / g starch to 31U / g starch, 32U / g starch to 33U / g starch, 34U / g starch to 35U / g starch, 36U / g starch to 37U / g starch, 38U / g starch to 39U / g starch, 40U / g starch to 41U / g starch, 42U / g starch to 43U / g starch, 44U / g starch to 45U / g starch, 46U / g starch to 47U / g starch, 48U / g starch to 49U / g starch, 50U / g starch to U / g starch ~ 11U / g starch, 11U / g starch ~ 12U / g starch, 12U / g starch ~ 13U / g starch, 13U / g starch ~ 14U / g starch, 14U / g starch ~ 15U / g starch, 15U / g starch ~ 16U / g starch, 16U / g starch ~ 17U / g starch, 17U / g starch ~ 18U / g starch, 18U / g starch ~ 19U / g starch or 19U / g starch ~ 20U / g starch.

[0087] In one aspect, in step (1), the liquefaction reaction is carried out simultaneously during the gelatinization process by adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, heating the suspension to 80-100° C., and maintaining the temperature for 20-60 min;

[0088] On the one hand, after the liquefaction reaction is carried out simultaneously during the gelatinization process, the DE value of the starch is controlled to be 2-8.

[0089] In one aspect, when the liquefying enzyme is α-amylase, the added amount is: calculated on the basis of substrate, 1U / g starch to 20U / g starch;

[0090] α-amylase activity determination method: DNS method was used to determine the reducing end content released by enzymatic starch for characterization. 0.3g soluble starch was added to 30mL PB buffer (NaH2PO4 / Na2HPO4, 0.2mM, pH 6.0), 1% soluble starch suspension was prepared, heated and boiled for 15min, cooled to room temperature, 1.8mL was transferred to a 2mL centrifuge tube, and then the centrifuge tube was placed in a 50℃ metal bath for preheating for 10min, 200μL of appropriately diluted enzyme solution was added, and oscillated at 300rpm. 200μL of the reaction solution was taken from the centrifuge tube with 800μL PB buffer and 1000μL DNS reagent added at 0min, 2min, 4min, 6min, 8min, and 10min, respectively, and reacted in a boiling water bath for 5min. After cooling in an ice water bath for 10min, the absorbance was measured at 540nm, and the enzyme activity was calculated by comparing with the glucose standard curve. 1U enzyme activity is defined as the amount of enzyme required to hydrolyze starch and release 1μmol of reducing sugar per minute at 50°C and pH 6.0.

[0091] On the one hand, in one embodiment of the present invention, when the liquefying enzyme is cyclodextrin glucosyltransferase, the added amount is: calculated on the basis of substrate, 1U / g starch to 20U / g starch.

[0092] In one aspect, the present invention starts the debranching reaction and the cyclization reaction after the starch suspension is liquefied.

[0093] In one aspect, the debranching reaction of starch of the present invention:

[0094] Starch debranching reaction refers to the process of removing branch points in amylopectin during the biosynthesis of starch to obtain straight-chain starch. This process is of great significance in starch metabolism and industrial application.

[0095] Factors that affect de-branching:

[0096] Structure of branching points: Starch is a polysaccharide composed of glucose units connected by α-1,4- and α-1,6-glycosidic bonds. Branched starches, such as glycogen and some types of starch, contain α-1,6-glycosidic bonds as branching points. Role of debranching enzymes: Debranching reactions are usually catalyzed by specific enzymes, which are called debranching enzymes (such as pullulanase, EC 3.2.1.41). These enzymes recognize α-1,6-glycosidic bonds and cut them off. Biological significance of debranching reactions: In plants, debranching reactions help regulate starch degradation and affect the final structure and properties of starch. In animals, debranching enzymes help break down starch during digestion. Industrial applications: In the food industry, debranching reactions can be used to produce amylose, which has different physical and chemical properties, such as higher solubility and viscosity stability. Amylose is widely used in food, textile, papermaking and other industries. Enzymatic debranching: In industrial production, starch debranching reactions can be carried out enzymatically, which has the advantages of mild reaction conditions, high efficiency and few by-products. Genetic engineering: Through genetic engineering, the activity of starch synthase can be changed to produce starch with different structures in plants, including starch with fewer branches. Regulation of debranching reactions: The efficiency and specificity of debranching reactions can be controlled by adjusting enzyme concentration, temperature, pH value and other conditions.

[0097] On the one hand, the debranching enzyme can be a pullulanase, which is a type of starch debranching enzyme named because it can specifically hydrolyze pullulan (Pullulan, a polymer of maltotriose connected by α-1,6 glycosidic bonds). It belongs to the amylase class and can specifically cut the α-1,6 glycosidic bonds in the branch points of starch, cut off the entire branch structure, and form straight-chain starch; the pullulanase can be a commercially available pullulanase, a pullulanase prepared by microbial fermentation, or a chemically synthesized pullulanase, and the method used can be any method known in the art.

[0098] Pullulanase activity determination method: Pullulanase activity was determined using the 3,5-dinitrosalicylic acid method (3,5-Dinitrosalicylicacid, DNS). Using 10 mg / ml glucose as the standard, a standard curve was prepared under different dilution conditions. 1 g of pullulan was dissolved in 100 mL of phosphate buffer (20 mM pH 6.0) to prepare a 1% (w / v) substrate. Take 0.9 ml of the substrate and preheat it at 40 ° C for 10 min, add 0.1 mL of enzyme solution (0.1 mg / mL), and after reacting for 10 min, add 1.0 mL of DNS to terminate the reaction. After the sample was colored in a boiling water bath for 5 min, it was immediately cooled in ice water. The absorbance was measured at 540 nm. The amount of enzyme required to catalyze the reduction power equivalent to 1 μmol of glucose per minute is defined as one enzyme activity unit (U).

[0099] In one aspect, the debranching enzyme can be an isoamylase, which only hydrolyzes the α-1,6 glycoside chain at the branch point of glycogen or amylopectin, cuts off the entire side branch, and forms amylose of varying lengths. The isoamylase can be a commercially available isoamylase, an isoamylase prepared by microbial fermentation, or a chemically synthesized isoamylase, and the method used can be any method known in the art.

[0100] On the one hand, the debranching enzyme can be a pullulanase, which is a general term for a class of enzymes that can catalyze the hydrolysis of α-1,6-glycosidic bonds in polysaccharide compounds such as starch to form straight-chain starch; the pullulanase can be a commercially available pullulanase, a pullulanase prepared by microbial fermentation, or a chemically synthesized pullulanase, and the method used can be any method known in the art.

[0101] In one aspect, the cyclization reaction is: the cyclization reaction of cyclodextrin refers to the process of forming cyclodextrin molecules on the basis of starch or its derivatives through the action of 4-α-glycosyltransferase (4αGT) or cyclodextrin Glucosyltransferase (CGTase). The mechanism of the cyclization reaction involves several key steps: the E region of CGTase has multiple maltose binding sites (MBS), which can bind to the substrate and guide the substrate into the active center of the enzyme.

[0102] In the cyclization process, amylose or maltose first binds to the MBS1 site of the enzyme, then extends to the MBS2 site, and is finally guided to the active center of the B domain of the enzyme for cyclization. Factors affecting the cyclodextrin cyclization reaction include the type and source of the enzyme, reaction time, temperature, pH value, enzyme dosage, additives, substrate type and concentration, and the degree of pretreatment of the raw materials. These factors affect not only the yield of cyclodextrin, but also the specificity of the product.

[0103] In one aspect, the cyclase can be a 4-alpha-glycosyltransferase, a cyclodextrin glucosyltransferase.

[0104] On the one hand, the 4-α-glycosyltransferase can be derived from: Deinococcus geothermalis, Aerococcus, Thermus filamentous, Escherichia coli, Thermus ancient, Thermus lettrichia, Thermus aquaticus, Streptococcus agalactiae, Corynebacterium glutamicum, Synechococcus PCC 6803, Synechococcus PCC 6804, Manihot esculenta, Arabidopsis thaliana, Solanum tuberosum.

[0105] On the one hand, the cyclodextrin glucosyltransferase can be a commercially available cyclodextrin glucosyltransferase, a cyclodextrin glucosyltransferase prepared by microbial fermentation, or a chemically synthesized cyclodextrin glucosyltransferase, and the method used can be any method known in the art.

[0106] In one aspect, in the debranching reaction, the amount of debranching enzyme added, calculated on the basis of substrate, is at least 100 U / g starch.

[0107] On the one hand, in the debranching reaction, the amount of debranching enzyme added can be 600U / g starch to 610U / g starch, can be 610U / g starch to 620U / g starch, can be 620U / g starch to 630U / g starch, can be 630U / g starch to 640U / g starch, can be 640U / g starch to 650U / g starch, can be 650U / g starch to 660U / g starch, can be 660U / g starch to 670U / g starch, can be 670U / g starch to 680U / g starch, can be 680U / g starch to 690U / g starch, can be 690U / g starch to 700U / g starch, can be It can be 700U / g starch to 710U / g starch, 710U / g starch to 720U / g starch, 720U / g starch to 730U / g starch, 730U / g starch to 740U / g starch, 740U / g starch to 750U / g starch, 750U / g starch to 760U / g starch, 760U / g starch to 770U / g starch, 770U / g starch to 780U / g starch, 780U / g starch to 790U / g starch, 790U / g starch to 800U / g starch, and 800U / g starch to 1000U / g starch.

[0108] On the one hand, in the cyclization reaction, the amount of cyclase added, calculated based on the substrate, can be: 1 U / g starch, 5 U / g starch, 10 U / g starch, 20 U / g starch, 100 U / g starch.

[0109] On the one hand, in the cyclization reaction, the amount of cyclase added can be 1 U / g starch, 2 U / g starch, 3 U / g starch, 4 U / g starch, 5 U / g starch, 6 U / g starch, 7 U / g starch, 8 U / g starch, 9 U / g starch, 10 U / g starch, 11 U / g starch, 12 U / g starch, 13 U / g starch, 14 U / g starch, 15 U / g starch, 16 U / g starch, 17 U / g starch, 18 U / g starch, 19 U / g starch, 20 U / g starch, 30 U / g starch, 40 U / g starch, 50 U / g starch, 60 U / g starch, 70 U / g starch, 80 U / g starch, 90 U / g starch or 100 U / g starch.

[0110] On the one hand, in the cyclization reaction, the amount of cyclase added can be 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 11U / g starch, 11U / g starch to 12U / g starch, 12U / g starch to 13U / g starch, 13U / g starch to 14U / g starch. powder, 14U / g starch ~ 15U / g starch, 15U / g starch ~ 16U / g starch, 16U / g starch ~ 17U / g starch, 17U / g starch ~ 18U / g starch, 18U / g starch ~ 19U / g starch, 19U / g starch ~ 20U / g starch, 20U / g starch ~ 30U / g starch, 30U / g starch ~ 40U / g, 40U / g starch ~ 50U / g, 50U / g starch ~ 60U / g, 60U / g starch ~ 70U / g, 70U / g starch ~ 80U / g, 80U / g starch ~ 90U / g or 90U / g starch ~ 100U / g.

[0111] On the one hand, the simultaneous reaction conditions for debranching and cyclization are set. In the present invention, the achievable reaction conditions for simultaneous debranching and cyclization can be: 50°C for 12 h; 50°C for 24 h; 60°C for 12 h; 60°C for 24 h, etc.

[0112] The conditions for synchronous debranching and cyclization are not limited to the above conditions.

[0113] In one aspect, the temperature for performing simultaneous debranching and cyclization can be 50°C to 80°C.

[0114] On the one hand, the temperature for simultaneous debranching and cyclization can be 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C.

[0115] In one aspect, the time for synchronous debranching and cyclization is 1 to 48 hours.

[0116] On the one hand, the time for synchronous debranching and cyclization is: 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h.

[0117] In one aspect, after the simultaneous debranching and cyclization, the present invention adds amylase to carry out purification reaction.

[0118] In one aspect, the present invention can also perform a debranching reaction first and then a cyclization reaction. The conditions for the debranching reaction and the cyclization reaction can be: 50° C. to 80° C.; and the time is: 1 to 48 hours.

[0119] In one aspect, the amylase can be a β-amylase, a saccharifying enzyme;

[0120] In one aspect, the β-amylase, also known as starch β-1,4-maltosidase, is a type of amylase, which is widely present in higher plants such as barley, wheat, sweet potato, soybean, and microorganisms such as Bacillus. The β-amylase can be commercially available β-amylase, β-amylase prepared by microbial fermentation, or chemically synthesized β-amylase, and the method used can be any method known in the art.

[0121] β-amylase activity determination method: β-amylase activity level was determined by 3,5-dinitrosalicylic acid method (DNS method): 0.94 mL of 1% soluble starch solution prepared with Na2HPO4-NaH2PO4 buffer (pH 6.0) was taken, preheated at 40°C for 5 min, 0.06 mL of β-amylase solution was added, mixed evenly, kept at 40°C for 15 min, 0.4 mL of reaction solution was added with 1.2 mL of DNS reagent, heated in a boiling water bath for 10 min, cooled to room temperature, 0.2 mL was added with 0.8 mL of deionized water, mixed evenly, and the OD value was measured at a wavelength of 540 nm. The standard curve was drawn with different concentrations of standard maltose solution as the X-axis and the OD value of the reaction solution measured at a wavelength of 540 nm as the Y-axis. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose soluble starch to produce 1 mg of maltose per hour under pH 6.0 and 40°C conditions.

[0122] On the one hand, the saccharifying enzyme is a customary name, and its scientific name is α-1,4-glucose hydrolase (α-1,4-Glucan glucohydrolace). The saccharifying enzyme can be a commercially available saccharifying enzyme, a saccharifying enzyme prepared by microbial fermentation, or a chemically synthesized saccharifying enzyme, and the method used can be any method known in the art.

[0123] In one aspect, the steps after amylase purification can be: alcohol precipitation treatment, dialysis;

[0124] In one aspect, the alcohol precipitation is performed using anhydrous ethanol.

[0125] In one aspect, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 1000 Da to remove small molecule sugars.

[0126] In one aspect, the drying method includes but is not limited to vacuum drying, freeze drying, spray drying, drum drying, fluidized bed drying, microwave drying, and ultrasonic drying.

[0127] In one aspect, any LR-CDs having suitable properties for use herein are purified, before or after any modification or conversion, by any method known in the art to remove impurities native to the starch or generated during processing.

[0128] On the one hand, the product of preparing LR-CDs from starch by enzyme catalysis contains not only LR-CDs, but also common cyclodextrins, chain dextrins and oligomaltosaccharides. Therefore, the first step in separating LR-CDs is to remove non-cyclic components. Since LR-CDs do not have reducing ends, they cannot be degraded by amylases that recognize the ends, such as β-amylase, isoamylase, and glucoamylase. Therefore, these enzymes are used to catalyze the LR-CDs product, which can degrade the chain molecules, thereby allowing the cyclic components to be initially purified.

[0129] On the one hand, the purification and separation method is thin layer chromatography, which is a commonly used separation method and is simple and easy to use. However, in the separation and analysis of sugars, conventional silica gel plates used in TLC are only used for the separation of monosaccharides and oligosaccharides.

[0130] On the one hand, the purification and separation method is high performance liquid chromatography (HPLC). The key factor in HPLC separation of LR-CDs is the selection of chromatographic columns and mobile phases. The LR-CDs and oligosaccharide mixture produced after starch catalysis needs to be separated by a gel column, and the LR-CDs component is collected and then separated by a reversed phase ODS column.

[0131] On the one hand, the purification of LR-CD is carried out by separation using chromatographic analysis technology. The purification can be carried out according to the method of Taira et al. (Taira H, Nagase H, Endo T, et al. Isolation, Purification and Characterization of Large-Ring Cyclodextrins (CD36-CD39) [J]. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 2006, 56 (1-2): 23-28) using a reverse chromatographic column and an amino column to separate CD36, CD37, CD38 and CD39 respectively; or according to the method of Koizumi et al. (Zheng MY, Endo T, Zimmermann W. Synthesis of large-ring cyclodextrins by cyclodextrin glucanotransferases from bacterial isolates [J]. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 2002, 44 (1-4): 387-390), the reaction product was eluted using an ODS column, and the methanol eluent was adjusted to different volume fractions to finally obtain LR-CDs products in the range of DP10-11, DP12-20 and DP21-31.

[0132] On the one hand, the addition amount of the amylase for purification can be 1U / g starch to 100U / g, and can be 1U / g starch, 2U / g starch, 3U / g starch, 4U / g starch, 5U / g starch, 6U / g starch, 7U / g starch, 8U / g starch, 9U / g starch, 10U / g starch, 11U / g starch, 12U / g starch, 13U / g starch, 14U / g starch, 15U / g starch, 16U / g starch, 17U / g starch, 18U / g starch, 19U / g starch, 20U / g starch, 21U / g starch, 22U / g starch, 23U / g starch, 24U / g starch, 25U / g starch, 26U / g starch, 27U / g starch, 28U / g starch, 29U / g starch or 30U / g starch.

[0133] On the one hand, in the purification reaction, the amount of amylase added can be 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 11U / g starch, 11U / g starch to 12U / g starch, 12U / g starch to 13U / g starch, 13U / g starch to 14U / g starch, 14U / g starch to 15U / g starch, 15U / g starch ~ 16U / g starch, 16U / g starch ~ 17U / g starch, 17U / g starch ~ 18U / g starch, 18U / g starch ~ 19U / g starch, 19U / g starch ~ 20U / g starch, 21U / g starch ~ 22U / g starch, 22U / g starch ~ 23U / g starch, 23U / g starch ~ 24U / g starch, 24U / g starch ~ 25U / g starch, 25U / g starch ~ 26U / g starch, 26U / g starch ~ 27U / g starch, 27U / g starch ~ 28U / g starch, 28U / g starch ~ 29U / g starch or 29U / g starch ~ 30U / g starch.

[0134] In one aspect, the reaction temperature of the purified amylase can be 30°C to 60°C.

[0135] In one aspect, the temperature of the purification reaction can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C.

[0136] In one aspect, the purification reaction time is: 1 to 48 hours.

[0137] On the one hand, the time of purification reaction is: 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h.

[0138] From the foregoing, it will be appreciated that the present disclosure may be embodied in various ways, including but not limited to the following:

[0139] Embodiment 1: A method for preparing LR-CDs, the method comprising: a) adding a liquefaction enzyme to a starch suspension to simultaneously carry out a liquefaction reaction during the gelatinization process; the mass fraction of starch in the starch suspension is greater than 7%; preferably greater than 10%; and more preferably greater than 20%; and b) after the reaction, adding a debranching enzyme and a cyclase to the reaction system at the same time for simultaneous action, or first adding a debranching enzyme to the obtained starch for debranching and then adding a cyclase for reaction; c) adding an amylase to the obtained reaction product for reaction, and then obtaining LR-CDs after a preliminary purification step.

[0140] Example 2: A method for preparing LR-CDs, the method comprising: first adding a liquefaction enzyme to liquefy the starch while gelatinizing it; after liquefaction is completed, using a debranching enzyme and a cyclase to simultaneously debranch and cyclize the starch; or first adding a debranching enzyme to the obtained starch for debranching, and then adding a cyclase to react; after the reaction is completed, inactivating the enzyme, centrifuging the starch, further purifying the supernatant, treating the starch with amylase, precipitating the starch with alcohol, centrifuging the starch, and dialyzing the starch, and then drying the starch to obtain the target product LR-CDs.

[0141] Example 3: The method described in Examples 1-2, wherein the starch is commercially available starch or chemically prepared starch, and the starch can be cereal starch, bean starch, potato starch, starch from other plant sources, etc., such as corn, peas, potatoes, sweet potatoes, barley, wheat, rice, cassava, sorghum, lotus root, and yam.

[0142] Embodiment 4: The method according to embodiments 1-3, wherein the starch can be reacted in the form of a starch suspension;

[0143] The starch suspension was prepared by accurately weighing potato starch (purchased from Hangzhou Prostar Starch Co., Ltd.), adding sodium citrate buffer (20 mmol / L, pH 6.0), placing in a 40-60° C. water bath, stirring at a speed of 100-200 rpm for 0-20 min to uniformly disperse the starch to form a starch suspension.

[0144] The mass fraction of starch in the starch suspension can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%.

[0145] Embodiment 5: The method according to embodiments 1-3, wherein the starch can be reacted in the form of a starch suspension, and the mass fraction of the starch in the starch suspension can be 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, 19% to 20%, 21% to 22%, 22% to 23% %, 23% to 24%, 24% to 25%, 25% to 26%, 26% to 27%, 27% to 28%, 28% to 29%, 29% to 30%, 31% to 32%, 32% to 33%, 33% to 34%, 34% to 35%, 35% to 36%, 36% to 37%, 37% to 38%, 38% to 39%, 39% to 40%, 40% to 41%, 41% to 42%, 42% to 43%, 43% to 44%, 44% to 45%, 45% to 46%, 46% to 47%, 47% to 48%, 48% to 49% or 49% to 50%.

[0146] Embodiment 6: The method according to embodiments 1-5, wherein the gelatinization is performed under certain temperature conditions after adding water; in the present invention, the starch suspension can be gelatinized, and the temperature can be directly increased for gelatinization; different types of starch have different gelatinization temperatures, for example, the gelatinization temperature of corn starch is approximately between 60°C and 80°C; the gelatinization temperature of potato starch is approximately between 60°C and 80°C; the gelatinization temperature of wheat starch is approximately between 60°C and 70°C; the gelatinization temperature of rice starch is approximately between 70°C and 85°C; the gelatinization temperature of lotus seed starch is approximately between 70°C and 80°C.

[0147] Example 7: The method according to Examples 1-6, wherein in the liquefaction reaction, the 4-α-glycosyltransferase can be derived from: Deinococcus geothermalis, Aerococcus, Thermus filamentous, Escherichia coli, Thermusancient, Thermus lettrichia, Thermus aquaticus, Streptococcus agalactiae, Corynebacterium glutamicum, Synechococcus PCC 6803, Synechococcus PCC 6804, Manihotesculenta, Arabidopsis thaliana, Solanum tuberosum.

[0148] Example 8: According to the method described in Examples 1-7, in the liquefaction reaction, the added amount of 4-α-glycosyltransferase can be 1U / g starch, 2U / g starch, 3U / g starch, 4U / g starch, 5U / g starch, 6U / g starch, 7U / g starch, 8U / g starch, 9U / g starch, 10U / g starch, 11U / g starch, 12U / g starch, 13U / g starch, 14U / g starch, 15U / g starch, 16U / g starch, 17U / g starch, 18U / g starch, 19U / g starch or 20U / g starch.

[0149] Embodiment 9: According to the method described in Embodiments 1-8, in the liquefaction reaction, the amount of 4-α-glycosyltransferase added can be 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 1 0U / g starch, 10U / g starch - 11U / g starch, 11U / g starch - 12U / g starch, 12U / g starch - 13U / g starch, 13U / g starch - 14U / g starch, 14U / g starch - 15U / g starch, 15U / g starch - 16U / g starch, 16U / g starch - 17U / g starch, 17U / g starch - 18U / g starch, 18U / g starch - 19U / g starch or 19U / g starch - 20U / g starch.

[0150] Example 10: According to the method described in Examples 1-9, in step (1), the liquefaction reaction is carried out simultaneously during the gelatinization process by adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, heating the starch suspension to 80-100°C and maintaining the temperature for 20-60 minutes;

[0151] Preferably, after the simultaneous liquefaction and gelatinization reactions, the DE value of the starch is controlled to be 2-8.

[0152] Embodiment 11: According to the method described in Embodiments 1-10, in step (1), the liquefaction reaction is carried out simultaneously during the gelatinization process by adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, heating the mixture to 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or 90°C and maintaining the mixture for 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, 65 min, 66 min, 67 min, 68 min, 69 min, 70 min, 71 min, 72 min, 73 min, 74 min, 75 min, 76 min, 77 min, 78 min, 79 min, 80 min, 81 min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45mi n, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min or 60min.

[0153] Example 12: According to the method described in Examples 1-11, the debranching enzyme can be pullulanase, and the pullulanase can be commercially available pullulanase, or a pullulanase prepared by microbial fermentation, or a chemically synthesized pullulanase. The method used can be any method known in the art.

[0154] Example 13: According to the method described in Examples 1-11, the debranching enzyme can be an isoamylase, the branched isoamylase can be a commercially available isoamylase, an isoamylase prepared by microbial fermentation, or a chemically synthesized isoamylase, and the method used can be any method known in the art.

[0155] Example 14: According to the method described in Examples 1-11, the debranching enzyme can be pullulanase, which can be commercially available pullulanase, pullulanase prepared by microbial fermentation, or chemically synthesized pullulanase. The method used can be any method known in the art.

[0156] Example 15: According to the method described in Examples 1-14, the cyclase can be a 4-α-glycosyltransferase or a cyclodextrin glucosyltransferase; the 4-α-glycosyltransferase can be derived from: Deinococcus geothermalis, Aerococcus, Thermus filamentous, Escherichia coli, Thermus ancient, Thermus lettrichia, Thermus aquaticus, Streptococcus agalactiae, Corynebacterium glutamicum, Synechococcus PCC 6803, Synechococcus PCC 6804, Manihot esculenta, Arabidopsis thaliana, Solanum tuberosum.

[0157] Example 16: According to the method described in Examples 1-15, the cyclodextrin glucosyltransferase can be a commercially available cyclodextrin glucosyltransferase, a cyclodextrin glucosyltransferase prepared by microbial fermentation, or a chemically synthesized cyclodextrin glucosyltransferase. The method used can be any method known in the art.

[0158] Example 17: According to the method described in Examples 1-16, in the debranching reaction, the amount of debranching enzyme added can be 600U / g starch to 610U / g starch, can be 610U / g starch to 620U / g starch, can be 620U / g starch to 630U / g starch, can be 630U / g starch to 640U / g starch, can be 640U / g starch to 650U / g starch, can be 650U / g starch to 660U / g starch, can be 660U / g starch to 670U / g starch, can be 670U / g starch to 680U / g starch, can be 680U / g starch to 690U / g starch, can be 690U / g starch to 70 0U / g starch, can be 700U / g starch - 710U / g starch, can be 710U / g starch - 720U / g starch, can be 720U / g starch - 730U / g starch, can be 730U / g starch - 740U / g starch, can be 740U / g starch - 750U / g starch, can be 750U / g starch - 760U / g starch, can be 760U / g starch - 770U / g starch, can be 770U / g starch - 780U / g starch, can be 780U / g starch - 790U / g starch, can be 790U / g starch - 800U / g starch, can be 800U / g starch - 1000U / g starch.

[0159] In the cyclization reaction, the amount of cyclase added can be 1 U / g starch, 2 U / g starch, 3 U / g starch, 4 U / g starch, 5 U / g starch, 6 U / g starch, 7 U / g starch, 8 U / g starch, 9 U / g starch, 10 U / g starch, 11 U / g starch, 12 U / g starch, 13 U / g starch, 14 U / g starch, 15 U / g starch, 16 U / g starch, 17 U / g starch, 18 U / g starch, 19 U / g starch, 20 U / g starch, 30 U / g starch, 40 U / g starch, 50 U / g starch, 60 U / g starch, 70 U / g starch, 80 U / g starch, 90 U / g starch or 100 U / g starch.

[0160] Or, in the cyclization reaction, the addition amount of the cyclase can be 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 11U / g starch, 11U / g starch to 12U / g starch, 12U / g starch to 13U / g starch, 13U / g starch to 14U / g starch, 14U / g starch ~ 15U / g starch, 15U / g starch ~ 16U / g starch, 16U / g starch ~ 17U / g starch, 17U / g starch ~ 18U / g starch, 18U / g starch ~ 19U / g starch, 19U / g starch ~ 20U / g starch, 20U / g starch ~ 30U / g starch, 30U / g starch ~ 40U / g, 40U / g starch ~ 50U / g, 50U / g starch ~ 60U / g, 60U / g starch ~ 70U / g, 70U / g starch ~ 80U / g, 80U / g starch ~ 90U / g or 90U / g starch ~ 100U / g.

[0161] Example 18: According to the method described in Examples 1-17, the temperature for performing debranching and cyclization synchronization can be 50° C. to 80° C. The time for debranching and cyclization synchronization is 1 to 48 hours.

[0162] Or first add a debranching enzyme to the obtained starch for debranching, and then add a cyclase to react; the temperature of the debranching or cyclization reaction can be 50°C to 80°C; the time of the debranching or cyclization reaction is 1 to 48 hours.

[0163] Example 19: According to the method described in Examples 1-18, the temperature of the simultaneous debranching and cyclization or the temperature of a separate reaction can be 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C. The time for synchronous debranching and cyclization or separate reaction is: 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h.

[0164] Example 20: According to the method described in Examples 1-19, the amylase may be β-amylase or saccharifying enzyme; the β-amylase may be commercially available β-amylase, or β-amylase prepared by microbial fermentation, or chemically synthesized β-amylase, and the method adopted may be any method known in the art.

[0165] The saccharifying enzyme may be a commercially available saccharifying enzyme, a saccharifying enzyme prepared by microbial fermentation, or a chemically synthesized saccharifying enzyme, and the method used may be any method known in the art.

[0166] Embodiment 21: According to the method described in Embodiments 1-20, the addition amount of the amylase can be 1U / g starch to 100U / g starch; preferably, it can be 1U / g starch, 2U / g starch, 3U / g starch, 4U / g starch, 5U / g starch, 6U / g starch, 7U / g starch, 8U / g starch, 9U / g starch, 10U / g starch, 11U / g starch, 12U / g starch, 13U / g starch, 14U / g starch, 15U / g starch, 16U / g starch, 17U / g starch, 18U / g starch, 19U / g starch, 20U / g starch, 21U / g starch, 22U / g starch, 23U / g starch, 24U / g starch, 25U / g starch, 26U / g starch, 27U / g starch, 28U / g starch, 29U / g starch or 30U / g starch. The temperature of the purification reaction can be any temperature between 30° C. and 60° C. The time of the purification reaction can be any time between 1 and 48 hours.

[0167] Embodiment 22: According to the method described in Embodiments 1-21, in the purification reaction, the amount of amylase added can be 1U / g starch to 100U / g starch; preferably, it can be 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 11U / g starch, 11U / g starch to 12U / g starch, 12U / g starch to 13U / g starch, 13U / g starch to 1 4U / g starch, 14U / g starch - 15U / g starch, 15U / g starch - 16U / g starch, 16U / g starch - 17U / g starch, 17U / g starch - 18U / g starch, 18U / g starch - 19U / g starch, 19U / g starch - 20U / g starch, 21U / g starch - 22U / g starch, 22U / g starch - 23U / g starch, 23U / g starch - 24U / g starch, 24U / g starch - 25U / g starch, 25U / g starch - 26U / g starch, 26U / g starch - 27U / g starch, 27U / g starch - 28U / g starch, 28U / g starch - 29U / g starch or 29U / g starch - 30U / g starch. The temperature of the purification reaction can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C. The time for the purification reaction is: 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h.

[0168] Example 23: According to the method described in Examples 1-22, the purification method is: any LR-CDs with suitable properties for use herein are purified by any method known in the prior art before or after any modification or conversion to remove impurities that are natural to the starch or generated during processing. The purification and separation method is thin layer chromatography, high performance liquid chromatography (HPLC), and separation using chromatographic analysis technology. Or the purification steps can be: alcohol precipitation treatment, dialysis, and drying; the alcohol precipitation is: alcohol precipitation using anhydrous ethanol. The dialysis is performed by dialysis using a dialysis bag with a molecular weight cutoff of 1000Da to remove small molecule sugars. The drying method includes but is not limited to hot air drying, vacuum drying, freeze drying, spray drying, drum drying, fluidized bed drying, microwave drying, and ultrasonic drying.

[0169] The following examples are presented for purposes of illustration and should not be construed as limiting.

[0170] Example 1: Preparation of LR-CDs under high substrate concentration conditions

[0171] The specific steps are as follows:

[0172] 1. Preparation of LR-CDs

[0173] (1) Preparation of high concentration starch

[0174] Accurately weigh 10 g of potato starch (purchased from Hangzhou Prostar Starch Co., Ltd.), add 23.3 mL of sodium citrate buffer (20 mmol / L, pH 6.0), place in a 50°C water bath, stir at 160 rpm for 10 min to evenly disperse it to form a starch suspension with a concentration of 30% (w / w).

[0175] (2) Gelatinization and liquefaction of high-concentration starch

[0176] Add 5 U / g starch of 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) to the 30% (w / w) starch suspension prepared in step (1), and place the suspension in a water bath;

[0177] The water bath temperature was set to 99.9°C. The starch was gelatinized and liquefied during the process of the water bath rising from 50°C to 99.9°C. After the reaction, the DE value of the starch was 2 to 4. The enzyme was then inactivated for 30 minutes.

[0178] (3) Synchronous action of pullulanase debranching and 4-α-glycosyltransferase cyclization

[0179] To the liquefied starch solution obtained in step (2), 700 U / g starch (excess) of pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41, CAS: 9075-68-7, purchased from Novozymes Denmark) and 10 U / g starch of 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) were added simultaneously. After reacting at 60° C. for 12 h, the enzyme was inactivated in a boiling water bath for 30 min, and the denatured enzyme protein was removed by centrifugation at 8000×g for 10 min, and the supernatant was collected.

[0180] (4) β-amylase treatment

[0181] Add 30 U / g starch of β-amylase (derived from sweet potato, EC3.2.1.2, 974 U / mg, purchased from Sigma-Aldrich, USA) to the supernatant collected after centrifugation in step (3), and react at 50°C for 12 h to remove unreacted linear dextrin. After the reaction is completed, inactivate the enzyme in a boiling water bath for 30 min. Centrifuge the reaction solution at 8000×g for 10 min to remove denatured enzyme protein, and collect the supernatant.

[0182] (5) Alcohol precipitation treatment

[0183] Add 4 volumes of anhydrous ethanol to the supernatant obtained after centrifugation in step (4), place in a 4°C refrigerator for alcohol precipitation for 12 hours, and centrifuge at 8000×g for 10 minutes to collect the precipitate.

[0184] (6) Removal of small molecule sugars by dialysis

[0185] 50 mL of deionized water was added to the precipitate obtained after centrifugation in step (5) to dissolve it, and dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove small molecule sugars.

[0186] (7) Freeze drying

[0187] The LR-CDs solution after dialyzation in step (6) was collected into a disposable plastic culture dish with a diameter of 90 mm and a height of 10 mm, and then frozen at -80°C for 6 h, and then freeze-dried for 48 h to obtain the LR-CDs sample.

[0188] 2. Identification of LR-CDs

[0189] (1) Distribution of degree of polymerization of LR-CDs

[0190] The degree of polymerization distribution of LR-CDs was determined by WATERS MALDI SYNAPT Q-TOF MS. The chromatographic column was BEHC182.1×150mmol / L1.7μm, and the detection wavelength was 200-400nm. The sample was prepared with ultrapure water at a concentration of 1mg / mL. The mobile phase was 0.1% formic acid (A) and acetonitrile (B), and the elution program was: 0-40min, 100% A; 40-45min, 70% A and 30% B; 45-50min, 20% A and 80% B; 50-55min, 100% B. The column temperature was 45℃, the injection volume was 5μL, and the flow rate was 0.3mL / min. The ionization mode was electrospray ionization-negative ion mode, and the ion spray voltage was 4kV. The nebulizing and drying gases were nitrogen, and the collision gas was helium.

[0191] LR-CDs were prepared using 4-α-glycosyltransferase using 10 g of potato starch as a substrate. After freeze-drying, 2378 mg of LR-CDs sample was collected, with a yield of 23.78%.

[0192] (2) Mass spectrometry identification

[0193] The mass spectrometry results of the samples can be found in Figure 1 ( Figure 1 In the figure, CA represents LR-CDs, the subscript represents the degree of polymerization of LR-CDs, and the calculation formula for the molecular weight of LR-CDs is: 162n, n represents the degree of polymerization of LR-CDs; the calculation formula for the molecular weight of linear dextrin with the corresponding degree of polymerization is: 162n+18).

[0194] The mass spectrometry results of the samples can be found in Figure 1 ( Figure 2 In the figure, CA represents LR-CDs, the subscript represents the degree of polymerization of LR-CDs, and the calculation formula of the molecular weight of LR-CDs is: 162n, where n represents the degree of polymerization of macrocyclodextrin).

[0195] Figure 1 is the mass spectrum of LR-CDs prepared by synchronous reaction of pullulanase and 4-α-glycosyltransferase for 12 hours. Figure 1 It can be seen that the prepared LR-CDs sample is a mixture of LR-CDs with different polymerization degrees. The purity of LR-CDs is relatively high, and no obvious linear dextrin molecular weight is doped. LR-CDs with a polymerization degree ranging from 9 to 36 can be prepared by the method of this embodiment.

[0196] Example 2: Effect of reaction time

[0197] The specific implementation method is the same as Example 1, except that the time for the synchronous reaction of pullulanase and 4-α-glycosyltransferase is changed from 12 h to 24 h.

[0198] After freeze-drying, 2742 mg of LR-CDs sample was collected, and the yield reached 27.42%.

[0199] The mass spectrometry results of the samples can be found in Figure 2 ( Figure 2 In the figure, CA represents LR-CDs, the subscript represents the degree of polymerization of LR-CDs, and the calculation formula of the molecular weight of LR-CDs is: 162n, where n represents the degree of polymerization of LR-CDs).

[0200] Depend on Figure 2 It can be seen that the LR-CDs with a degree of polymerization ranging from 9 to 19 prepared by the synchronous reaction of pullulanase and 4-α-glycosyltransferase for 24 hours also contained some linear dextrins in the freeze-dried samples collected at the end. This may be because 4-α-glycosyltransferase itself has a coupling activity opposite to the cyclization activity. In the later stage of the 4-α-glycosyltransferase reaction, this activity will cause the already generated LR-CDs to be ring-opened and degraded to form linear dextrins. β-amylase does not completely degrade more linear dextrins, resulting in a decrease in the yield of actual LR-CDs in the prepared samples.

[0201] Example 3: Effect of addition of α-amylase

[0202] The specific implementation method is the same as Example 1, except that the 4-α-glycosyltransferase used for liquefaction in step (2) is replaced with α-amylase (derived from Bacillus licheniformis, EC 3.2.1.1, CAS: 9000-90-2, purchased from Novozymes Denmark, 7704U / mL). The water bath temperature is set to 99.9°C. The gelatinization and liquefaction of starch are achieved in the process of the water bath rising from 50°C to 99.9°C. After the reaction is completed, the DE value of the starch is 2 to 4. Then keep the enzyme inactivated for 30 minutes. The rest of the operations are consistent with Example 1.

[0203] According to the method of Example 1, 802.5 mg of product was prepared with a yield of 8.03%. α-Amylase is an endoenzyme that randomly cuts α-1,4 glycosidic bonds from inside starch molecules, causing the viscosity to drop rapidly and present a dilute solution state. Starch is hydrolyzed into short-chain dextrins and a small amount of oligosaccharides. These small molecular weight oligosaccharide hydrolysis products are not effective substrates for 4-α-glycosyltransferase, resulting in a reduction in the final LR-CDs yield.

[0204] Example 4: Effects of 4-α-glycosyltransferases from different sources

[0205] The specific steps are as follows:

[0206] 1. Preparation of LR-CDs

[0207] (1) Preparation of high concentration starch:

[0208] Accurately weigh 10 g of potato starch (purchased from Hangzhou Prostar Starch Co., Ltd.), add 23.3 mL of sodium citrate buffer (20 mmol / L, pH 6.0), place in a 50°C water bath, stir at 160 rpm for 10 min to evenly disperse it to form a starch suspension with a concentration of 30% (w / w).

[0209] (2) Gelatinization and liquefaction of high-concentration starch:

[0210] 5U / g starch 4-α-glycosyltransferase (derived from: Pyrobaculum arsenaticum DSM13514, Pyrobaculum oguniense TE7, Pyrobaculumferrireducens, uncultured Acidilobus sp.MG, uncultured Acidilobus sp.OSP8, NCBI numbers: ABP49821.1, AFA40300.1, AET33957.1, ESQ23201.1, ESQ23782.1) was added to the 30% (w / w) starch suspension prepared in step (1), and then placed in a water bath; the water bath temperature was set to 99.9°C, and the water bath was heated from 50°C to 99.9°C to achieve gelatinization and liquefaction of the starch, and the DE value was controlled to be 2-4. Then the enzyme was inactivated for 30 minutes.

[0211] (3) Synchronous action of pullulanase debranching and 4-α-glycosyltransferase cyclization:

[0212] To the liquefied starch solution obtained in step (2), 700 U / g starch (excess) of pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41 purchased from Novozymes Denmark) and 10 U / g starch of 4-α-glycosyltransferase were added simultaneously. The 4-α-glycosyltransferase (the same as the enzyme in step (2)) was derived from: Pyrobaculum arsenaticum DSM13514, Pyrobaculum oguniense TE7, Pyrobaculumferrireducens, uncultured Acidilobus sp.MG, uncultured Acidilobus sp.OSP8, NCBI numbers are: ABP49821.1, AFA40300.1, AET33957.1, ESQ23201.1, ESQ23782.1 (sequence similarities with WP_013679179.1 are 65%, 65%, 60%, 59%, 59%, respectively). After reacting at 60°C for 12 hours, the enzyme was inactivated in a boiling water bath for 30 minutes, and the denatured enzyme protein was removed by centrifugation at 8000×g for 10 minutes, and the supernatant was collected.

[0213] (4) β-amylase treatment:

[0214] Add 30 U / g starch of β-amylase (derived from sweet potato, EC3.2.1.2, 974 U / mg, purchased from Sigma-Aldrich, USA) to the supernatant collected after centrifugation in step (3), and react at 50°C for 12 h to remove unreacted linear dextrin. After the reaction is completed, inactivate the enzyme in a boiling water bath for 30 min. Centrifuge the reaction solution at 8000×g for 10 min to remove denatured enzyme protein, and collect the supernatant.

[0215] (5) Alcohol precipitation treatment:

[0216] Add 4 volumes of anhydrous ethanol to the supernatant obtained after centrifugation in step (4), place in a 4°C refrigerator for alcohol precipitation for 12 hours, and centrifuge at 8000×g for 10 minutes to collect the precipitate.

[0217] (6) Removal of small molecule sugars by dialysis:

[0218] 50 mL of deionized water was added to the precipitate obtained after centrifugation in step (5) to dissolve it, and dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove small molecule sugars.

[0219] (7) Freeze drying:

[0220] The LR-CDs solution after dialyzation in step (6) was collected into a disposable plastic culture dish with a diameter of 90 mm and a height of 10 mm, and then frozen at -80°C for 6 h, and then freeze-dried for 48 h to obtain the LR-CDs sample.

[0221] After freeze-drying, LR-CDs were prepared with 4-α-glycosyltransferases from 5 different sources (derived from: Pyrobaculum arsenaticum DSM13514, Pyrobaculum oguniense TE7, Pyrobaculum ferrireducens, uncultured Acidilobus sp.MG, uncultured Acidilobus sp.OSP8), with a yield of 15% to 30%. Compared with the 4-α-glycosyltransferase from Thermoproteus uzoniensis, the yield of enzymes from other sources may be slightly reduced, which may be due to the different optimal temperatures and temperature stability of 4-α-glycosyltransferases from different sources. In the preparation process of the present invention, the reaction temperature during cyclization is 60°C. If the temperature is too low, the problem of starch aging and retrogradation will occur during the reaction process, and the utilization rate of the starch substrate will decrease; if the temperature is too high, the enzyme activity of pullulanase will be affected.

[0222] Example 5: Effect of different starch substrate concentrations

[0223] The specific steps are as follows:

[0224] 1. Preparation of LR-CDs

[0225] (1) Preparation of high concentration starch:

[0226] Accurately weigh potato native starch (purchased from Hangzhou Prostar Starch Co., Ltd.), add sodium citrate buffer (20 mmol / L, pH 6.0), and stir in a 50°C water bath at a speed of 160 rpm for 10 min to make it evenly dispersed, and prepare starch suspensions with starch substrate concentrations of 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), and 50% (w / w), respectively.

[0227] (2) Gelatinization and liquefaction of high-concentration starch:

[0228] Add 5 U / g starch of 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) to the 30% (w / w) starch suspension prepared in step (1), and place the suspension in a water bath;

[0229] The temperature of the water bath was set to 99.9°C. The starch was gelatinized and liquefied during the process of the water bath rising from 50°C to 99.9°C, and the DE value was controlled to be 2 to 4. The enzyme was then inactivated for 30 minutes.

[0230] (3) Synchronous action of pullulanase debranching and 4-α-glycosyltransferase cyclization:

[0231] To the liquefied starch solution obtained in step (2), 700 U / g starch (excess) of pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41 purchased from Novozymes Denmark) and 10 U / g starch of 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) were added simultaneously. After reacting at 60° C. for 12 h, the enzyme was inactivated by boiling water bath for 30 min, and the denatured enzyme protein was removed by centrifugation at 8000×g for 10 min, and the supernatant was collected.

[0232] (4) β-amylase treatment:

[0233] Add 30 U / g starch of β-amylase (derived from sweet potato, EC3.2.1.2, 974 U / mg, purchased from Sigma-Aldrich, USA) to the supernatant collected after centrifugation in step (3), and react at 50°C for 12 h to remove unreacted linear dextrin. After the reaction is completed, inactivate the enzyme in a boiling water bath for 30 min. Centrifuge the reaction solution at 8000×g for 10 min to remove denatured enzyme protein, and collect the supernatant.

[0234] (5) Alcohol precipitation treatment:

[0235] Add 4 times the volume of anhydrous ethanol to the supernatant obtained after centrifugation in step (4), place in a 4°C refrigerator for alcohol precipitation for 12 hours, and centrifuge at 8000×g for 10 minutes to collect the precipitate.

[0236] (6) Removal of small molecule sugars by dialysis:

[0237] 50 mL of deionized water was added to the precipitate obtained after centrifugation in step (5) to dissolve it, and dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove small molecule sugars.

[0238] (7) Freeze drying:

[0239] The LR-CDs solution after dialyzation in step (6) was collected into a disposable plastic culture dish with a diameter of 90 mm and a height of 10 mm, and then frozen at -80°C for 6 h, and then freeze-dried for 48 h to obtain the LR-CDs sample.

[0240] After freeze-drying, the starch suspensions with substrate concentrations of 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), and 50% (w / w) were finally prepared to 2648 mg, 2435 mg, 2378 mg, 1757 mg, and 1563 mg, respectively, and the calculated yields were 26.48%, 24.35%, 23.78%, 17.57%, and 15.63%, respectively. It can be seen from the results that as the concentration of the starch substrate increases, the yield of the starch substrate with a substrate concentration of 40% and 50% decreases. This may be because the starch substrate concentration is too high. During the gelatinization and liquefaction process, the starch molecules are difficult to absorb water and swell, and the liquefaction effect of 4-α-glycosyltransferase is not good, which reduces the accessibility between the subsequent enzyme and the substrate, resulting in a decrease in the final yield.

[0241] Example 6: Effects of different types of starch substrates

[0242] The specific steps are as follows:

[0243] (1) Preparation of high concentration starch:

[0244] Accurately weigh 10 g of pea starch, tapioca starch, corn starch, glutinous corn starch, and rice starch, add 23.3 mL of sodium citrate buffer (20 mmol / L, pH 6.0), place in a 50° C. water bath, stir at 160 rpm for 10 min to evenly disperse, and form starch suspensions with a concentration of 30% (w / w).

[0245] (2) Gelatinization and liquefaction of high-concentration starch:

[0246] Add 5 U / g starch of 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) to the 30% (w / w) starch suspension prepared in step (1), and place the suspension in a water bath;

[0247] The temperature of the water bath was set to 99.9°C. The starch was gelatinized and liquefied during the process of the water bath rising from 50°C to 99.9°C, and the DE value was controlled to be 2 to 4. The enzyme was then inactivated for 30 minutes.

[0248] (3) Synchronous action of pullulanase debranching and 4-α-glycosyltransferase cyclization:

[0249] To the liquefied starch solution obtained in step (2), 700 U / g starch (excess) of pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41 purchased from Novozymes Denmark) and 10 U / g starch of 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) were added simultaneously. After reacting at 60° C. for 12 h, the enzyme was inactivated by boiling water bath for 30 min, and the denatured enzyme protein was removed by centrifugation at 8000×g for 10 min, and the supernatant was collected.

[0250] (4) β-amylase treatment:

[0251] Add 30 U / g starch of β-amylase (derived from sweet potato, EC3.2.1.2, 974 U / mg, purchased from Sigma-Aldrich, USA) to the supernatant collected after centrifugation in step (3), and react at 50°C for 12 h to remove unreacted linear dextrin. After the reaction is completed, inactivate the enzyme in a boiling water bath for 30 min. Centrifuge the reaction solution at 8000×g for 10 min to remove denatured enzyme protein, and collect the supernatant.

[0252] (5) Alcohol precipitation treatment:

[0253] Add 4 volumes of anhydrous ethanol to the supernatant obtained after centrifugation in step (4), place in a 4°C refrigerator for alcohol precipitation for 12 hours, and centrifuge at 8000×g for 10 minutes to collect the precipitate.

[0254] (6) Removal of small molecule sugars by dialysis:

[0255] 50 mL of deionized water was added to the precipitate obtained after centrifugation in step (5) to dissolve it, and dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove small molecule sugars.

[0256] (7) Freeze drying:

[0257] The LR-CDs solution after dialyzation in step (6) was collected into a disposable plastic culture dish with a diameter of 90 mm and a height of 10 mm, and then frozen at -80°C for 6 h, and then freeze-dried for 48 h to obtain the LR-CDs sample.

[0258] After freeze-drying, 2891 mg, 2134 mg, 2257 mg, 1868 mg and 2098 mg of samples were collected (corresponding to pea starch, tapioca starch, corn starch, glutinous corn starch and rice starch, respectively), with yields of 28.91%, 21.34%, 22.57%, 18.68% and 20.98%, respectively.

[0259] LR-CDs products were prepared using different types of starch substrates, among which pea starch had the highest yield. The reason may be that pea starch contains a higher content of amylose, which increases the accessibility of 4-α-glycosyltransferase.

[0260] Example 7: Different enzyme addition amounts

[0261] The following different addition amounts are merely demonstrations of different embodiments and cannot limit the protection scope of the present invention.

[0262] The specific steps are as follows:

[0263] 1. Amount of 4-α-glycosyltransferase added

[0264] Specific implementation method: The same as Example 1, after adding 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) to the 30% (w / w) starch suspension prepared in step (1), the suspension was placed in a water bath; the water bath temperature was set to 99.9°C, and the gelatinization and liquefaction of the starch were achieved in the process of the water bath rising from 50°C to 99.9°C, and the DE value was controlled to be 2-4. The enzyme was then kept for 30 minutes to inactivate the enzyme.

[0265] The difference is that, in the adjustment step (2), the added amount of the 4-α-glycosyltransferase, calculated based on the substrate, can be: 0.1U / g starch, 0.5U / g starch, 1U / g starch, 2U / g starch, 5U / g starch, 10U / g, 20U / g starch.

[0266] Other conditions were in accordance with the method of Example 1, and the results showed that:

[0267] With different added amounts of enzyme, samples of 1061 mg, 1457 mg, 1536 mg, 1843 mg, 2378 mg, 2018 mg and 1613 mg were collected (the corresponding added amounts were 0.1 U / g starch, 0.5 U / g starch, 1 U / g starch, 2 U / g starch, 5 U / g starch, 10 U / g and 20 U / g starch, respectively), and the yields reached 10.61%, 14.57%, 15.36%, 18.43%, 23.78%, 20.18% and 16.13%.

[0268] The results are as follows Figure 3 As shown, if the amount of 4-α-glycosyltransferase added is too small, the liquefaction of the high-concentration starch substrate cannot be achieved, and the yield is low; if the amount of 4-α-glycosyltransferase added is too much, the starch substrate will be fully hydrolyzed and cannot serve as an effective substrate for subsequent cyclization, and the yield will also be reduced.

[0269] 2. The amount of pullulanase added:

[0270] The specific implementation is the same as that of Example 1, except that the adjustment step (3) is:

[0271] Pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41 purchased from Novozymes Denmark) and 10 U / g starch 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) were added to the liquefied starch solution obtained in step (2) at the same time. After reacting at 60° C. for 12 h, the enzyme was inactivated in a boiling water bath for 30 min, and the denatured enzyme protein was removed by centrifugation at 8000×g for 10 min, and the supernatant was collected.

[0272] The added amount of the pullulanase, calculated based on the substrate, is: 1U / g starch, 10U / g starch, 50U / g starch, 100U / g starch, 500U / g starch, 700U / g, and 1000U / g starch.

[0273] Other conditions were in accordance with the method of Example 1, and the results showed that:

[0274] With different added amounts of enzyme, 892 mg, 1218 mg, 1697 mg, 1963 mg, 2351 mg, 2378 mg and 2413 mg of samples were collected, and the yields reached 8.92%, 12.18%, 16.97%, 19.63%, 23.51%, 23.78% and 24.13% (the corresponding added amounts were: 1 U / g starch, 10 U / g starch, 50 U / g starch, 100 U / g starch, 500 U / g starch, 700 U / g and 1000 U / g starch).

[0275] The results are as follows Figure 4 As shown, the amount of pullulanase added is too small to achieve complete debranching of the high-concentration starch substrate to provide an effective substrate for 4-α-glycosyltransferase, resulting in a low yield; after the amount of pullulanase added reaches an excessive amount, the yield increases and gradually remains stable.

[0276] 3. Amount of glycosyltransferase added

[0277] The specific implementation is the same as that of Example 1, except that the adjustment step (3) is:

[0278] To the liquefied starch solution obtained in step (2), 700 U / g starch (excess) of pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41, CAS: 9075-68-7, purchased from Novozymes Denmark) and 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) were added simultaneously. After reacting at 60° C. for 12 h, the enzyme was inactivated by boiling water bath for 30 min, and the denatured enzyme protein was removed by centrifugation at 8000×g for 10 min, and the supernatant was collected.

[0279] The added amount of the 4-α-glycosyltransferase, calculated based on the substrate, can be: 1U / g starch, 3U / g starch, 5U / g starch, 8U / g starch, 10U / g starch, 50U / g starch, 100U / g starch.

[0280] Other conditions were in accordance with the method of Example 1, and the results showed that:

[0281] With different added amounts of enzyme, samples of 539 mg, 1308 mg, 1923 mg, 2137 mg, 2378 mg, 1882 mg and 1411 mg were collected, with yields of 5.39%, 13.08%, 19.23%, 21.37%, 23.78%, 18.82% and 14.11% (the corresponding enzyme addition amounts were: 1U / g starch, 3U / g starch, 5U / g starch, 8U / g starch, 10U / g starch, 50U / g starch and 100U / g starch, respectively).

[0282] The results are as follows Figure 5 As shown in the figure, with the increase of enzyme addition, the yield of LR-CDs also increases. When the enzyme addition amount is 10U / g, the yield of LR-CDs reaches the maximum, which is 23.78%. After that, with the increase of enzyme addition, the yield of LR-CDs shows a significant downward trend, and when the enzyme addition amount is 100U / g, the yield of LR-CDs is only 14.11%. This shows that increasing the amount of enzyme addition can increase the yield of LR-CDs, but when the amount of enzyme addition exceeds a certain amount, increasing the amount of enzyme addition will reduce the yield. The possible reason is that if the enzyme addition amount is too much, the coupling activity of 4-α-glycosyltransferase may degrade LR-CDs, resulting in a decrease in yield, and too much enzyme protein will cause the reaction system to be contaminated.

[0283] 4. The amount of β-amylase added:

[0284] The specific implementation is the same as that of Example 1, except that the adjustment step (4) is:

[0285] Add 30 U / g starch of β-amylase (derived from sweet potato, EC3.2.1.2, 974 U / mg, purchased from Sigma-Aldrich, USA) to the supernatant collected after centrifugation in step (3), and react at 50°C for 12 h to remove unreacted linear dextrin. After the reaction is completed, inactivate the enzyme in a boiling water bath for 30 min. Centrifuge the reaction solution at 8000×g for 10 min to remove denatured enzyme protein, and collect the supernatant.

[0286] The added amount of the β-amylase, calculated based on the substrate, can be: 1U / g starch, 10U / g starch, 20U / g starch, 30U / g starch, 40U / g starch, 50U / g, 100U / g starch.

[0287] Other conditions were in accordance with the method of Example 1, and the results showed that:

[0288] With different added amounts of enzyme, 3129 mg, 2981 mg, 2669 mg, 2327 mg, 2351 mg, 2427 mg and 2313 mg of samples were collected, with yields of 31.29%, 29.81%, 26.69%, 23.78%, 23.51%, 23.27% and 23.13% (the corresponding enzyme addition amounts were: 1 U / g starch, 10 U / g starch, 20 U / g starch, 30 U / g starch, 40 U / g starch, 50 U / g and 100 U / g starch, respectively).

[0289] The results are as follows Figure 6 As shown, the amount of β-amylase added is too small to completely degrade the linear dextrin mixed in the product, resulting in an increase in the yield after freeze-drying; when the amount of β-amylase added reaches 30U / g starch, the linear dextrin is completely degraded into disaccharides and removed during the subsequent dialysis process, and the yield gradually remains stable.

[0290] Example 8: Effects of pullulanase and 4-α-glycosyltransferase after sequential reactions

[0291] The specific implementation method is the same as Example 1, except that in step (3) of step 1, debranching and cyclization are performed in separate steps, that is, step (3) is:

[0292] Add 700 U / g starch (excess) of pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41 purchased from Novozymes Denmark) to the liquefied starch solution obtained in step (2), react at 60° C. for 12 h, and then inactivate the enzyme;

[0293] To the enzyme-inactivated solution, add 10 U / g starch 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1), react at 60°C for 12 h, inactivate the enzyme in a boiling water bath for 30 min, centrifuge at 8000×g for 10 min to remove the denatured enzyme protein, and collect the supernatant.

[0294] According to the method of Example 1, 2471 mg of product was prepared with a yield of 24.71%.

[0295] The results showed that the principles of distributed reaction, debranching before cyclization and synchronous reaction were consistent. In the synchronous reaction, pullulanase was required to complete debranching before 4-α-glycosyltransferase could carry out the cyclization reaction. Therefore, debranching before cyclization and step-by-step reaction had little effect on the yield.

[0296] The following examples are presented for comparison purposes:

[0297] Comparative Example 1:

[0298] The specific implementation method is the same as Example 1, except that in step (2) of step 1, 4-α-glycosyltransferase is not added, that is, step (2) is adjusted to:

[0299] The 30% (w / w) starch suspension prepared in step (1) is placed in a water bath, and the temperature of the water bath is set to 99.9° C. When the temperature of the water bath rises from 50° C. to 99.9° C., the liquefaction step is omitted.

[0300] According to the method of Example 1, 639 mg of product was prepared with a yield of 6.39%.

[0301] The results showed that after omitting the 4-α-glycosyltransferase liquefaction step, the viscosity of the high-concentration starch suspension increased rapidly under heating conditions, forming large lumps of incompletely gelatinized starch. In the subsequent enzyme reaction, the accessibility between the enzyme and the starch substrate was inhibited, the yield was greatly reduced, only part of the starch substrate reacted, and after the reaction was completed, the starch was still in lumps.

[0302] Comparative Example 2

[0303] The specific implementation method is the same as Example 1, except that, in step (3) of step 1, debranching and cyclization are performed in separate steps, first cyclization and then debranching, that is, step (3) is:

[0304] Add 10 U / g starch of 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) to the liquefied starch solution obtained in step (2), react at 75° C. for 12 h, and then inactivate the enzyme;

[0305] 700 U / g starch (excess) of pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41 purchased from Novozymes Denmark) was added to the solution after enzyme inactivation. After reaction at 60°C for 12 h, the enzyme was inactivated in a boiling water bath for 30 min, and the denatured enzyme protein was removed by centrifugation at 8000 × g for 10 min, and the supernatant was collected.

[0306] 543 mg of product was prepared according to the method of Example 1, with a yield of 5.43%.

[0307] This is because 4-α-glycosyltransferase has a high substrate specificity, and the yield of LR-CDs obtained by its action on amylopectin is very low or cannot be obtained. Therefore, natural starch is not suitable as a substrate for preparing LR-CDs.

[0308] In summary, the present invention provides a process for preparing LR-CDs from a high-concentration substrate, which greatly improves the production efficiency of LR-CDs and has a high purity of the product, which makes it possible to prepare LR-CDs in large quantities and realize their industrial production and application.

[0309] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing macrocyclodextrins (LR-CDs), characterized in that: The method comprises the following steps: (1) adding a liquefaction enzyme to a starch suspension to simultaneously carry out a liquefaction reaction during the gelatinization process; the mass fraction (w / w) of starch in the starch suspension is greater than 7%, preferably greater than 10%, and more preferably greater than 20%; (2) adding a debranching enzyme and a cyclase to the starch obtained in step (1) at the same time, and reacting them to obtain a reaction product; or first adding a debranching enzyme to the starch obtained in step (1) to carry out a debranching reaction, and then adding a cyclase to carry out a reaction to obtain a reaction product; (3) Adding amylase to the reaction product obtained in step (2) to carry out reaction to prepare LR-CDs.

2. The method according to claim 1, characterized in that The mass fraction (w / w) of starch in the starch suspension is 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, 19% to 20%, 21% to 22%, 22% to 23%, 23% to 24%, 24% to 25%, 25% to 26%, 26% to 27%, 27% to 28%, 28% to 29%, 30% to 31%, 31% to 32%, 32% to 33%, 33% to 34%, 35% to 36%, 37% to 38%, 39% to 40%, 40% to 41%, 41% to 42%, 42% to 43%, 44% to 45%, 45% to 46%, 47% to 48%, 49% to 50%, 50% to 51%, 51% to 52%, 52% to 53%, 53% to 54%, 54% to 55%, 56% to 57%, 58% to 59%, 60% to 61%, 61% to 62%, 62% to 63%, 64% to 64%, 65% to 66%, 67% to 68%, 69% to 70%, 70% to 71%, 71% to 72%, 72% to 73%, 73% to 74%, 75% to 76%, 77% to 78%, 79% to 44%-45%, 45%-46%, 46%-47%, 47%-48%, 48%-49% or 49%-50%.

3. The method according to claim 1 or 2, characterized in that: In step (1), the liquefaction enzyme includes but is not limited to: α-amylase, 4-α-glycosyltransferase, cyclodextrin glucosyltransferase; when the liquefaction enzyme is 4-α-glycosyltransferase, the addition amount is: calculated on the substrate, 1U / g starch to 20U / g starch; when the liquefaction enzyme is α-amylase, the addition amount is: calculated on the substrate, 1U / g starch to 20U / g starch; when the liquefaction enzyme is cyclodextrin glucosyltransferase, the addition amount is: calculated on the substrate, 1U / g starch to 20U / g starch; In step (1), the liquefaction reaction is carried out simultaneously during the gelatinization process by adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, heating the suspension to 80-100° C., and maintaining the temperature for 20-60 min; Preferably, after the liquefaction reaction is carried out simultaneously during the gelatinization process, the DE value of the starch is controlled to be 2 to 8; Preferably, the addition amount of the liquefaction enzyme is: 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 1 1U / g starch, 11U / g starch to 12U / g starch, 12U / g starch to 13U / g starch, 13U / g starch to 14U / g starch, 14U / g starch to 15U / g starch, 15U / g starch to 16U / g starch, 16U / g starch to 17U / g starch, 17U / g starch to 18U / g starch, 18U / g starch to 19U / g starch or 19U / g starch to 20U / g starch; Preferably, the liquefaction enzyme is a 4-α-glycosyltransferase, and the 4-α-glycosyltransferase can be derived from: Deinococcus geothermalis, Aerococcus, Thermus filamentous, Escherichia coli, Thermus ancient, Thermus lettrichia, Thermus aquaticus, Streptococcus agalactiae, Corynebacterium glutamicum, Synechococcus PCC 6803, Synechococcus PCC 6804, Manihotesculenta, Arabidopsis thaliana, Solanum tuberosum.

4. The method according to any one of claims 1 to 3, characterized in that: In step (1), the liquefaction reaction is carried out simultaneously during the gelatinization process, and after adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, the temperature is raised to 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or 90°C; the holding time is 10-20min, 20-30min, 30-40min, 40-50min, 50-60min, 60-70min, 70-80min, 80-90min, 90-100min or 110-120min.

5. The method according to any one of claims 1 to 4, characterized in that: The debranching enzyme includes but is not limited to: pullulanase, isoamylase, pullulanase; the cyclase includes but is not limited to: 4-α-glycosyltransferase, cyclodextrin glucosyltransferase.

6. The method according to any one of claims 1 to 5, characterized in that: The amount of the debranching enzyme added is, based on the substrate, at least 100 U / g of starch; the amount of the cyclase added is: based on the substrate, 1 U / g starch to 100 U / g starch; Preferably, the addition amount of the cyclase is: 1U / g starch to 2U / g starch, 2U / g starch to 3U / g starch, 3U / g starch to 4U / g starch, 4U / g starch to 5U / g starch, 5U / g starch to 6U / g starch, 6U / g starch to 7U / g starch, 7U / g starch to 8U / g starch, 8U / g starch to 9U / g starch, 9U / g starch to 10U / g starch, 10U / g starch to 11U / g starch, 11U / g starch to 12U / g starch, 12U / g starch to 13U / g starch, 13U / g starch to 14U / g starch, 14U / g starch to 15U / g starch, 16U / g starch to 17U / g starch, 17U / g starch to 18U / g starch, 19U / g starch to 20U / g starch, 21U / g starch to 22U / g starch, 23U / g starch to 24U / g starch, 25U / g starch to 26U / g starch, 27U / g starch to 28U / g starch, 29U / g starch to 30U / g starch, 31U / g starch to 32U / g starch, 33U / g starch to 34U / g starch, 35U / g starch to 36U / g starch, 37U / g starch to 38U / g starch, 39U / g starch to 40U / g starch, 41U / g starch to 42U / g starch, 43U / g starch to 44U / g starch, 45U / g starch to 46U / g starch, 47U / g starch to 48U / g starch, 49U / g starch to 50U / g starch, U / g starch ~ 15U / g starch, 15U / g starch ~ 16U / g starch, 16U / g starch ~ 17U / g starch, 17U / g starch ~ 18U / g starch, 18U / g starch ~ 19U / g starch, 19U / g starch ~ 20U / g starch, 20U / g starch ~ 30U / g starch, 30U / g starch ~ 40U / g, 40U / g starch ~ 50U / g, 50U / g starch ~ 60U / g, 60U / g starch ~ 70U / g, 70U / g starch ~ 80U / g, 80U / g starch ~ 90U / g or 90U / g starch ~ 100U / g.

7. The method according to any one of claims 1 to 6, characterized in that: In step (2), the reaction conditions are: reaction at 50°C to 80°C for 1 to 48 hours; Preferably, the temperature is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C or 80°C; The reaction time is 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h or 48h.

8. The method according to any one of claims 1 to 7, characterized in that: In step (3), the amylase is β-amylase or saccharifying enzyme; the amount of the amylase added is, calculated on the basis of the substrate, 1U / g starch to 100U / g starch; the reaction temperature after adding the amylase is 30°C to 60°C, and the reaction time is 1 to 48h; Preferably, the temperature is 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C; The time is 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h or 48h.

9. The method according to any one of claims 1 to 8, characterized in that: Step (3) also includes a purification and separation step, and the purification and separation method is to obtain the product after alcohol precipitation, dialysis and drying; preferably, the purification and separation method includes but is not limited to: membrane separation, column separation, and multi-stage chromatography separation.

10. Use of the method according to any one of claims 1 to 9 or the LR-CDs prepared by the method according to any one of claims 1 to 9 in the preparation of food, biological products and pharmaceuticals.

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