Method for improving freeze-thaw stability of starch gel

Abstract

The invention discloses a method for improving freeze-thaw stability of starch gel, and belongs to the technical field of starch processing. Macrocyclodextrin (LR-CDs) is prepared by taking starch as a substrate and utilizing 4-alpha-glycosyl transferase, the LR-CDs are added into the starch to participate in a gelatinization process, and the starch gel is formed by cooling after gelatinization. The gel is subjected to multiple freeze-thaw cycles, and the water bleeding rate of the starch gel added with the LR-CDs is lower than that of the original starch gel, so that the freeze-thaw stability of the starch gel is improved. The method belongs to addition of homologous substances, is green and environment-friendly, and does not bring potential safety hazards. Starch is used as a main raw material of quick-frozen wheaten food, the freeze-thaw stability of the starch directly affects the quality of the product, and the method is of great significance to quick-frozen food processing.

Description

Technical Field

[0001] The invention relates to a method for improving the freeze-thaw stability of starch gel, and belongs to the technical field of starch processing. Background Art

[0002] Starch is widely used in various foods as an important food processing raw material and excellent additive. When starch-based foods are frozen, phase separation occurs due to the formation of ice crystals in the food matrix. After thawing, water can easily flow out of the dense network system, resulting in deterioration phenomena such as water loss and product hardening, which affects the overall sensory quality and shelf life of the product. In order to maintain the final quality of starch-based products, gels must be able to withstand adverse physical changes during freezing and thawing (freeze-thaw).

[0003] During the freezing process of starch gel, the water molecules bound by the three-dimensional network structure of the gel freeze to form ice crystals and detach from the gel system. When the frozen starch gel melts at room temperature, the ice crystals formed easily detach from the three-dimensional network structure of the starch gel. Repeated freeze-thaw treatments will increase the number of water molecules in the network structure and thus increase the structure of the ice crystals, aggravating the damage of the network structure by the ice crystals, thereby accelerating the separation between the water molecules and the gel network structure. This process is the dehydration of starch gel, also known as dehydration.

[0004] The freeze-thaw stability of starch directly affects the quality of quick-frozen pasta. There are some means to improve the freeze-thaw stability of quick-frozen pasta in the process of industrial production and processing. Different chemical reagents and modification methods can affect the freeze-thaw stability of starch to varying degrees. Deetae et al. treated rice starch with 1% sodium trimetaphosphate cross-linking treatment, 4% sodium tripolyphosphate cross-linking treatment, and a combination of the two to perform secondary denaturation treatment on starch. The results showed that the dehydration shrinkage rate of starch gel was reduced compared with the first two types of modified starch treated separately, showing better freeze-thaw stability. The possible mechanism is that the charge effect between the phosphate groups improves the stability of the starch structure, reduces the mechanical damage during the freeze-thaw process, and slows down the aging of starch and the precipitation of water molecules through the steric hindrance effect. Ye Jiangping physically modified the rice starch of frozen rice products by improved extrusion technology (IECT) and found that IECT treatment reduced the water separation rate of rice starch and delayed the change of the honeycomb network structure, effectively improving the stability of rice starch gel during repeated freeze-thaw cycles. In the previous research of this laboratory, Wang Yu used 4-α-glycosyltransferase from Thermoproteus uzoniensis to modify potato starch, and the freeze-thaw stability of the modified potato starch was improved. In addition, exogenous additives such as proteins, salts, low molecular weight sugars, polysaccharides, etc. can effectively reduce the starch aging rate and water loss rate by changing the movement and distribution of water molecules and interacting with starch molecules, thereby improving the freeze-thaw stability of starch. Summary of the invention

[0005] The inventor prepared LR-CDs and added them to starch for gelatinization to prepare a starch gel with improved freeze-thaw stability. The preparation process of the invention is efficient, the equipment is simple, and the product effect is good. The prepared starch gel is applied to quick-frozen food to improve its freeze-thaw stability and improve the quality of quick-frozen food, and has excellent application value.

[0006] The invention provides a method for improving the freeze-thaw stability of starch gel. The method comprises the following steps: dispersing starch in water to prepare a starch suspension, adding LR-CDs with a DP range of 10 to 50 to the starch suspension for gelatinization, cooling the suspension to room temperature, and forming a gel at 4°C, wherein the water separation rate of the starch gel during several freeze-thaw cycles is reduced, and the freeze-thaw stability is improved.

[0007] In one embodiment of the present invention, the degree of polymerization of the LR-CDs includes DPX, where X is any value between 9 and 50, such as DP9, DP10, and the like.

[0008] In one embodiment of the present invention, the degree of polymerization of the LR-CDs includes DPA-B, A is any value between 9 and 49, B is greater than A, and B is any value between 10 and 50.

[0009] In one embodiment of the present invention, the DP of the LR-CDs may be 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27 ~28, 28~29, 29~30, 30~31, 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.

[0010] In one embodiment of the present invention, the mass fraction of starch in the starch suspension is 1-30%; optionally, the mass fraction of starch in the starch suspension is 5-30% (w / w). Alternatively, 5-20% (w / w); alternatively, 5-15% (w / w); alternatively, 5-10% (w / w); alternatively, 65 (w / w); further optionally, 7% (w / w); optionally, the mass fraction of starch in the starch suspension is 1%-2% (w / w), 2%-3% (w / w), 3%-4% (w / w), 4%-5% (w / w), 5%-6% (w / w), 6%-7% (w / w), 7%-8% (w / w), 8%-9% (w / w), 9%-10% (w / w), 10%-11% (w / w), 11%-12% (w / w), 1 2%~13% (w / w), 13%~14% (w / w), 14%~15% (w / w), 15%~16% (w / w), 16%~17% (w / w), 17%~18% (w / w), 18%~19% (w / w), 19%~20% (w / w), 21%~22% (w / w), 22% to 23% (w / w), 23% to 24% (w / w), 24% to 25% (w / w), 25% to 26% (w / w), 26% to 27% (w / w), 27% to 28% (w / w), 28% to 29% (w / w) or 29% to 30% (w / w).

[0011] In one embodiment of the present invention, the mass fraction (w / w) of starch in the starch suspension is 5 to 30%.

[0012] In one embodiment of the present invention, the amount of LR-CDs added, calculated as the amount of starch added, is 1% to 20% (w / w) of the dry weight of starch. Alternatively, 2 to 10% (w / w); alternatively, 2 to 8% (w / w); alternatively, 3 to 7% (w / w); alternatively, 4 to 6% (w / w); further alternatively, 5% (w / w).

[0013] In one embodiment of the present invention, the addition amount of the macrocyclodextrin (LR-CDs), calculated as the addition amount of starch, is 1% to 2% (w / w), 2% to 3% (w / w), 3% to 4% (w / w), 4% to 5% (w / w), 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w), 15% to 16% (w / w), 16% to 17% (w / w), 17% to 18% (w / w), 18% to 19% (w / w) or 19% to 20% (w / w) of the dry weight of starch.

[0014] In one embodiment of the present invention, the gelatinization conditions are: 90-100° C., heating for 30-60 min to completely gelatinize the starch.

[0015] In one embodiment of the present invention, the gelatinization includes but is not limited to RVA instrument gelatinization and water bath gelatinization.

[0016] In one embodiment of the present invention, the starch includes but is not limited to potato starch, wheat starch, corn starch, and tapioca starch;

[0017] In one embodiment of the present invention, the gelatinization specifically comprises the following steps:

[0018] (1) After adding LR-CDs to the starch suspension, the mixture was kept at 45-55°C for 1-5 min, then heated to 90-100°C at a heating rate of 10-15°C / min, and kept at 90-100°C for 1-5 min;

[0019] (2) The sample obtained in step (1) is cooled to 45-55° C. at a rate of 10-15° C. / min and kept warm for 1-5 min; the entire gelatinization process needs to be stirred. Within 1-30 s of the initial gelatinization, the stirring paddle is stirred at a speed of 500-1000 r / min. After 1-30 s of the gelatinization process, the rotation speed is adjusted to 100-200 r / min.

[0020] In one embodiment of the present invention, the freeze-thaw cycle conditions are: freezing the gel sample at -80 to -20°C for 8 to 24 hours, and then thawing it in a water bath at 10 to 30°C for 1 to 5 hours, which is recorded as one freeze-thaw cycle.

[0021] In one embodiment of the present invention, the preparation method of LR-CDs is to gelatinize starch and then add a debranching enzyme to debranch, add a glycosyltransferase to cyclize after debranching, and add amylase to purify after cyclization to prepare LR-CDs.

[0022] 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 3.2.1.68).

[0023] 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;

[0024] In one embodiment of the present invention, the glycosyltransferase includes but is not limited to: 4-α-glycosyltransferase (EC 2.4.1.25) and cyclodextrin glucosyltransferase (EC 2.4.1.19).

[0025] In one embodiment of the present invention, the NCBI accession number of the 4-α-glycosyltransferase includes but is not limited to WP_013679179.1, ABP49821.1, AFA40300.1, AET33957.1, ESQ23201.1, and ESQ23782.1.

[0026] In one embodiment of the present invention, the cyclization conditions are: adding 1 to 100 U / g starch glycosyltransferase, and enzymolysis at 40 to 80° C. for 1 to 12 hours;

[0027] In one embodiment of the present invention, the amylase includes but is not limited to: β-amylase (EC 3.2.1.2) and saccharifying enzyme (EC 3.2.1.3).

[0028] 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;

[0029] 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;

[0030] In one embodiment of the present invention, anhydrous ethanol is used for the alcohol precipitation, and the ratio of the reaction solution to the ethanol addition is 1:1 to 1:10.

[0031] In one embodiment of the present invention, the molecular weight cut-off of the dialysis bag is 500 to 3000 Da.

[0032] In one embodiment of the present invention, the drying method includes freeze drying, atmospheric pressure drying, spray drying, drum drying or microwave drying.

[0033] In one embodiment of the present invention, the starch includes but is not limited to potato starch, corn starch, tapioca starch, and pea starch.

[0034] The present invention also provides LR-CDs prepared by the above method, wherein the DP range of the LR-CDs is 9-50.

[0035] The present invention also provides another method for preparing LR-CDs, which comprises the following steps:

[0036] (1) 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%;

[0037] (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;

[0038] (3) adding amylase to the reaction product obtained in step (2) to react and prepare LR-CDs;

[0039] In one embodiment of the present invention, the mass fraction of starch in the starch suspension is 7% to 50%;

[0040] In one embodiment of the present invention, in step (1), the liquefying enzyme includes but is not limited to: α-amylase, 4-α-glycosyltransferase, cyclodextrin glucosyltransferase; when the liquefying enzyme is 4-α-glycosyltransferase, the added amount is: calculated on the substrate, 1U / g starch to 20U / g starch; when the liquefying enzyme is α-amylase, the added amount is: calculated on the substrate, 1U / g starch to 20U / g starch; when the liquefying enzyme is cyclodextrin glucosyltransferase, the added amount is: calculated on the substrate, 1U / g starch to 20U / g starch.

[0041] In one embodiment of the present invention, 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 80-100° C. and maintaining the temperature for 20-60 min;

[0042] In one embodiment of the present invention, after the liquefaction reaction is carried out simultaneously during the gelatinization process, the DE value of the starch is controlled to be 2-8.

[0043] In one embodiment of the present invention, the addition amount of the liquefaction enzyme is: 1U / g starch to 20U / g starch;

[0044] In one embodiment of the present invention, the liquefying enzyme is 4-α-glycosyltransferase; 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; the amount of the debranching enzyme added is, based on the substrate, adding at least 100U / g starch debranching enzyme; the amount of the cyclase added is: calculated on the substrate, 1U / g starch to 20U / g starch;

[0045] 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;

[0046] 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.

[0047] In one embodiment of the present invention, the mass fraction of starch in the starch suspension is 1% to 2% (w / w), 2% to 3% (w / w), 3% to 4% (w / w), 4% to 5% (w / w), 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w), 15% ~ 16% (w / w), 16% ~ 17% (w / w), 17% ~ 18% (w / w), 18% ~ 19% (w / w), 19% ~ 20% (w / w), 21% ~ 22% (w / w), 22% ~ 23% (w / w), 23% to 24% (w / w), 24% to 25% (w / w), 25% to 26% (w / w), 26% to 27% (w / w), 27% to 28% (w / w), 28% to 29% (w / w) or 29% to 30% (w / w);

[0048] In one embodiment of the present invention, the addition amount of the macrocyclodextrin (LR-CDs), calculated as the addition amount of starch, is 1% to 2% (w / w), 2% to 3% (w / w), 3% to 4% (w / w), 4% to 5% (w / w), 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w), 15% to 16% (w / w), 16% to 17% (w / w), 17% to 18% (w / w), 18% to 19% (w / w) or 19% to 20% (w / w) of the dry weight of starch.

[0049] The present invention also provides a starch gel prepared by the method; the starch gel has the following properties: after cooling and standing for a period of time, the starch gel is subjected to 5 freeze-thaw cycle experiments, and the water separation rate of each freeze-thaw cycle is lower than that of the original starch gel.

[0050] The present invention also provides an application of the preparation method or the starch gel or LR-CDs with a polymerization degree DP ranging from 9 to 50 in improving the freeze-thaw stability of frozen food.

[0051] In one embodiment of the present invention, the frozen food includes but is not limited to frozen pasta, frozen ready-to-eat food, frozen dessert, and frozen pet food.

[0052] In one embodiment of the present invention, the frozen pasta mainly includes the following categories:

[0053] Frozen cooked noodles: Noodles are quickly frozen after being cooked and can be stored for a long time in the frozen state. After thawing, they can be eaten with simple conditioning.

[0054] Frozen raw noodles: Fresh wet noodles are quickly frozen after being made and refrigerated in a frozen state.

[0055] Frozen prepared noodles: After the noodles are cooked, soup and seasonings are added or they are fully prepared and then quickly frozen. They can be refrigerated for a long time in a frozen state.

[0056] In addition, there are some specific frozen pasta products, such as: quick-frozen pasta: including but not limited to steamed buns, steamed bread, dumplings, etc. These products are usually made from wheat flour and other raw materials, and are frozen after processing for easy consumption.

[0057] Quick-frozen udon noodles: A type of frozen cooked noodles, it is a traditional Japanese food that is preserved for a long time by being quickly frozen and can be eaten after simple conditioning after thawing.

[0058] Quick-frozen ready-to-eat meals: specifically refers to products made from two or more fresh agricultural, aquatic, and livestock products as raw materials, such as quick-frozen dumplings, glutinous rice balls, and rice dumplings. These products also belong to the category of frozen ready-to-eat meals.

[0059] Beneficial Effects

[0060] The invention provides a method for preparing LR-CDs by using 4-α-glycosyltransferase and application of the LR-CDs in preparing a starch gel with excellent freeze-thaw stability. After adding LR-CDs and gelatinizing with potato starch to form a gel, after several freeze-thaw cycles, the water separation rate of the starch gel added with LR-CDs is lower than that of the original starch gel, indicating that the starch gel has good freeze-thaw stability.

[0061] The method provided by the present invention belongs to adding homologous substances, is easy to operate, and will not make the food taste too sweet or too oily or make the texture of the food change unexpectedly like adding exogenous substances such as salt, sugar, oil, colloid, etc., nor will it have safety hazards like chemical modification. Starch is the main raw material of quick-frozen pasta, and its freeze-thaw stability directly affects the product quality. Therefore, improving the freeze-thaw stability of starch gel is of great significance in the processing of quick-frozen food. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 : LC-MS identification chart of the LR-CDs used.

[0063] Figure 2 : SEM images of starch gel before and after adding LR-CDs. DETAILED DESCRIPTION

[0064] 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.

[0065] Technical terms:

[0066] Large ring cyclodextrins: 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-glycosyltransferase (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.

[0067] Freeze-thaw stability: Freeze-thaw stability refers to the ability of a substance to maintain its physical and chemical properties unchanged after undergoing freezing and thawing processes. This concept is very important in many fields, especially in pharmaceuticals, biological products, food industry and materials science. As one of the most effective means of food preservation, freezing technology can effectively extend the shelf life of food, reduce water activity, and slow down the rate of chemical and enzymatic reactions. Frozen food will be accompanied by a series of temperature fluctuations during production, storage, transportation, sales and final consumption. For example, factors such as refrigerator differences, loading and unloading of goods, and defrosting of supermarket freezers cause frozen food to undergo multiple freeze-thaw processes, which causes the physical and chemical properties of frozen food to deteriorate and have an adverse effect on the shelf life of the exhibits. Starch is the main component of starch-based foods, so the freeze-thaw stability of starch gel directly affects the quality of starch-based frozen foods. During the low-temperature freezing process, the water in the frozen food will freeze into ice, resulting in phase separation. After thawing, the ice crystals melt into water and can be easily released from the food matrix, which is called syneresis.

[0068] It should be noted that the present invention can adopt any method to prepare the target product LR-CDs; the preparation method of the LR-CDs can be any of the following methods:

[0069] a. After gelatinizing the starch, 4-α-glycosyltransferase is added to carry out liquefaction reaction. After the reaction is completed, debranching enzyme and cyclase are added simultaneously for synchronous action. Amylase is added to the obtained reaction product for purification reaction, and LR-CDs are obtained after a purification step.

[0070] b. 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, then add glycosyltransferase to react, add amylase to the obtained reaction product for purification reaction, and obtain LR-CDs after purification steps.

[0071] c. Starch is used as raw material. After being debranched by debranching enzyme, an appropriate amount of glycosyltransferase is added, and the appropriate amount and action time are selected to produce LR-CDs. After the enzyme is inactivated by centrifugation, the starch is treated with amylase for a period of time, and then the enzyme is inactivated, centrifuged, rotary evaporated, alcohol precipitated and dialyzed. Finally, the target product LR-CDs is obtained by drying.

[0072] The target product LR-CDs can be prepared by any method as long as the DP is greater than 8 to achieve the technical solution of the present invention.

[0073] In one aspect, the DP of the LR-CDs ranges from 9-50.

[0074] In one aspect, the LR-CDs can have a DP of 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.

[0075] In one aspect, the DP of the LR-CDs can be 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 29-30, 31-32, 33-34, 35-36, 37-38, 39-40, 41-42, 43-44, 45-46, 47-48, 49-50, 50-51, 52-53, 54-55, 56-57, 58-59, 59-60, 61-61, 62-63, 64-64, 65-65 8-29, 29-30, 30-31, 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.

[0076] The following are just two examples of preparation methods of LR-CDs:

[0077] The first method for preparing LR-CDs:

[0078] The method for preparing LR-CDs of the present invention comprises the following steps: using starch as a raw material, after gelatinization, first adding a debranching enzyme to carry out a debranching reaction, and then carrying out secondary gelatinization; after the secondary gelatinization, adding an appropriate amount of glycosyltransferase to carry out a cyclization reaction; selecting a suitable addition amount and action time to produce LR-CDs; inactivating the enzyme by centrifugation; then carrying out amylase treatment for a period of time, inactivating the enzyme, centrifuging, rotary evaporating, alcohol precipitation and dialysis; and finally drying to obtain the target product LR-CDs.

[0079] 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 characteristics 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.

[0080] 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).

[0081] 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 1% to 10%.

[0082] On the one hand, 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%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0083] 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% 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% or 10% to 11%.

[0084] 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.

[0085] 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.

[0086] In one aspect, the debranching reaction of starch of the present invention:

[0087] 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.

[0088] Factors that affect de-branching:

[0089] 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.

[0090] 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.

[0091] Determination of pullulanase activity

[0092] The pullulanase activity was determined by 3,5-dinitrosalicylic acid (DNS) method. 10 mg / ml glucose was used as the standard and a standard curve was prepared under different dilution conditions.

[0093] Dissolve 1g of pullulan in 100mL of phosphate buffer (20mM pH 6.0) to prepare a 1% (w / v) substrate. Take 0.9ml of substrate and preheat it at 40℃ for 10min, add 0.1mL of enzyme solution (0.1mg / mL), react for 10min, and then add 1.0mL of DNS to terminate the reaction. After the sample is colored in a boiling water bath for 5min, it is immediately cooled in ice water. Measure the absorbance at 540nm. The amount of enzyme required to catalyze the production of reducing power equivalent to 1μmol of glucose per minute is defined as one enzyme activity unit (U).

[0094] In one aspect, the debranching enzyme can be an isoamylase, which only hydrolyzes the -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.

[0095] In one aspect, in the debranching reaction, the amount of debranching enzyme added, calculated on the basis of substrate, is at least 10 U / g starch.

[0096] 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.

[0097] In one aspect, the temperature at which the debranching reaction is carried out can be 40°C to 80°C.

[0098] In one aspect, the temperature at which the debranching reaction is carried out can be 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, 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.

[0099] In one aspect, the debranching reaction is carried out for 1 to 24 hours.

[0100] On the one hand, the time for carrying out the debranching 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.

[0101] The cyclization reaction is:

[0102] 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 cyclodextrin Glucosyltransferase (CGTase). The mechanism of 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.

[0103] On the one hand, during 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, pretreatment degree of raw materials, etc. These factors affect not only the yield of cyclodextrin, but also the specificity of the product.

[0104] In one aspect, the cyclase can be a 4-alpha-glycosyltransferase, a cyclodextrin glucosyltransferase.

[0105] 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 PCC6803, PCC 6804, Manihot esculenta, Arabidopsis thaliana, Solanum tuberosum.

[0106] In one aspect, the cyclodextrin glucosyltransferase may be a commercially available cyclodextrin glucosyltransferase, a cyclodextrin glucosyltransferase prepared by microbial fermentation, or a chemically synthesized cyclodextrin glucosyltransferase, and the method used may be any method known in the art.

[0107] On the one hand, in the cyclization reaction, the amount of cyclase added, calculated based on the substrate, can be: 1U / g starch, 2U / g starch, 5U / g starch, 10U / g starch, 20U / g starch, 30U / g starch, 40U / g starch, 50U / g starch, 60U / g starch, 70U / g starch, 80U / g starch, 90U / g starch, 100U / g starch.

[0108] 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 or 20 U / g starch.

[0109] In one aspect, 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 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 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, 51U / g starch to 52U / g starch, 53U / g starch to 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 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.

[0110] In one aspect, the temperature at which the cyclization reaction is carried out can be between 40°C and 80°C.

[0111] In one aspect, the temperature for conducting the cyclization reaction can be 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, 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.

[0112] In one aspect, the cyclization reaction is carried out for 1 to 12 hours.

[0113] In one aspect, the cyclization reaction is carried out for a period of time of 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h.

[0114] In the present invention, after the debranching reaction and the cyclization reaction are carried out, amylase is added to carry out the purification reaction.

[0115] In one aspect, the amylase can be a β-amylase, a saccharifying enzyme;

[0116] In one aspect, the β-amylase, also known as amylo-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 a commercially available β-amylase, a β-amylase prepared by microbial fermentation, or a chemically synthesized β-amylase, and the method used can be any method known in the art.

[0117] β-Amylase Activity Assay

[0118] The activity level of β-amylase 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 the conditions of pH 6.0 and 40°C.

[0119] 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.

[0120] In one aspect, the purification steps can be: alcohol precipitation, dialysis, and drying.

[0121] In one aspect, the alcohol precipitation is performed using anhydrous ethanol.

[0122] In one aspect, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 1000 Da to remove small molecule sugars.

[0123] In one aspect, the drying method includes but is not limited to natural drying, hot air drying, vacuum drying, freeze drying, spray drying, drum drying, fluidized bed drying, infrared drying, microwave drying, and ultrasonic drying.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] On the one hand, the purification of LR-CD is carried out by separation using chromatographic analysis technology. The purification method 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.

[0129] 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.

[0130] 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.

[0131] In one aspect, the reaction temperature of the purified amylase can be 40°C to 80°C.

[0132] In one aspect, the temperature of the purification reaction can be 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, 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.

[0133] In one aspect, the purification reaction time is: 1 to 24 hours.

[0134] On the one hand, the time of the 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.

[0135] The second method for preparing LR-CDs:

[0136] The present invention also provides a method for preparing large cyclodextrin LR-CDs, which comprises gelatinizing starch with high substrate concentration as a substrate, adding 4-α-glycosyltransferase to carry out a liquefaction reaction, adding a debranching enzyme and a cyclase to act synchronously after the reaction is completed, adding amylase to the obtained reaction product to carry out a purification reaction, and obtaining LR-CDs after a purification step.

[0137] 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.

[0138] 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%.

[0139] 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%.

[0140] In one aspect, the present invention adopts the method of first subjecting the gelatinized starch to a liquefaction reaction;

[0141] 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, 19U / g starch ~ 20U / g starch.

[0142] 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;

[0143] 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.

[0144] 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.

[0145] In one aspect, when the liquefying enzyme is cyclodextrin glucosyltransferase, the added amount is: calculated on the basis of substrate, 1U / g starch to 20U / g starch.

[0146] In one aspect, the present invention starts the debranching reaction and the cyclization reaction after the starch suspension is liquefied.

[0147] The enzymes and amounts added for the debranching reaction and the cyclization reaction are the same as those for the first preparation method, except that, in the second method, the debranching and cyclization are simultaneous reactions.

[0148] In the present invention, the 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.

[0149] The conditions for synchronous debranching and cyclization are not limited to the above conditions.

[0150] In one aspect, the temperature for performing simultaneous debranching and cyclization can be 50°C to 80°C.

[0151] 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.

[0152] In one aspect, the time for synchronous debranching and cyclization is 1 to 48 hours.

[0153] 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.

[0154] In one aspect, the present invention adds amylase to carry out purification reaction after the simultaneous debranching and cyclization; the method of purification reaction is the same as the first preparation method.

[0155] The invention provides a method for improving the freeze-thaw stability of starch gel. The method comprises the following steps: dispersing starch in water to prepare a starch suspension of a certain concentration, adding macrocyclodextrins (LR-CDs) with a degree of polymerization (DP) of 10 to 50 to the starch suspension for gelatinization, obtaining a starch gel after gelatinization, reducing the water separation rate of the starch gel during several freeze-thaw cycles, and improving the freeze-thaw stability.

[0156] In one aspect, the DP of the LR-CDs ranges from 9-50.

[0157] In one aspect, the LR-CDs can have a DP of 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.

[0158] In one aspect, the DP of the LR-CDs can be 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 29-30, 31-32, 33-34, 35-36, 37-38, 39-40, 41-42, 43-44, 45-46, 47-48, 49-50, 50-51, 52-53, 54-55, 56-57, 58-59, 59-60, 61-61, 62-63, 64-64, 65-65 8-29, 29-30, 30-31, 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.

[0159] In one aspect, the mass fraction of starch in the starch suspension is 1 to 30% (w / w).

[0160] In one aspect, the mass fraction of starch in the starch suspension can be 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 11% (w / w), 12% (w / w), 13% (w / w), 14% (w / w), 15 % (w / w), 16% (w / w), 17% (w / w), 18% (w / w), 19% (w / w), 20% (w / w), 21% (w / w), 22% (w / w), 2 3% (w / w), 24% (w / w), 25% (w / w), 26% (w / w), 27% (w / w), 28% (w / w), 29% (w / w) or 30% (w / w).

[0161] In one aspect, the mass fraction of starch in the starch suspension can be 1% to 2% (w / w), 2% to 3% (w / w), 3% to 4% (w / w), 4% to 5% (w / w), 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w). w), 15% ~ 16% (w / w), 16% ~ 17% (w / w), 17% ~ 18% (w / w), 18% ~ 19% (w / w), 19% ~ 20% (w / w), 21% ~ 22% (w / w), 22% ~ 23% (w / w ), 23% to 24% (w / w), 24% to 25% (w / w), 25% to 26% (w / w), 26% to 27% (w / w), 27% to 28% (w / w), 28% to 29% (w / w) or 29% to 30% (w / w).

[0162] 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.

[0163] 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.

[0164] In one aspect, the addition amount of the macrocyclodextrins (LR-CDs), based on the addition amount of starch, is 1% to 20% (w / w) of the dry weight of starch.

[0165] On the one hand, the amount of macrocyclodextrins (LR-CDs) added, based on the amount of starch added, is 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 11% (w / w), 12% (w / w), 13% (w / w), 14% (w / w), 15% (w / w), 16% (w / w), 17% (w / w), 18% (w / w), 19% (w / w) or 20% (w / w) of the dry weight of starch.

[0166] On the one hand, the addition amount of macrocyclodextrins (LR-CDs), based on the addition amount of starch, is 1% to 2% (w / w), 2% to 3% (w / w), 3% to 4% (w / w), 4% to 5% (w / w), 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w), 15% to 16% (w / w), 16% to 17% (w / w), 17% to 18% (w / w), 18% to 19% (w / w) or 19% to 20% (w / w) of the dry weight of starch.

[0167] In one aspect, the gelatinization reaction is to add macrocyclodextrins (LR-CDs) to a starch suspension, then heat the mixture to 80-100° C. and maintain the temperature for 20-60 min;

[0168] 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.

[0169] From the foregoing, it will be appreciated that the present disclosure may be embodied in various ways, including but not limited to the following:

[0170] Example 1: A method for improving the freeze-thaw stability of starch gel, the method comprising dispersing starch in water to form a starch suspension of a certain concentration, adding macrocyclodextrins (LR-CDs) with a degree of polymerization (DP) ranging from 10 to 50 to the starch suspension and then gelatinizing the starch suspension to obtain a starch gel after gelatinization, wherein the water separation rate of the starch gel decreases during several freeze-thaw cycles, and the freeze-thaw stability is improved.

[0171] Example 2: According to the method described in Example 1, the preparation method of the LR-CDs is: The preparation method of the LR-CDs can be any of the following methods:

[0172] a. After gelatinizing the starch, 4-α-glycosyltransferase is added to carry out liquefaction reaction. After the reaction is completed, debranching enzyme and cyclase are added simultaneously for synchronous action. Amylase is added to the obtained reaction product for purification reaction, and LR-CDs are obtained after a purification step.

[0173] b. 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, then add glycosyltransferase to react, add amylase to the obtained reaction product for purification reaction, and obtain LR-CDs after purification steps.

[0174] c. Starch is used as raw material. After being debranched by debranching enzyme, an appropriate amount of glycosyltransferase is added, and the appropriate amount and action time are selected to produce LR-CDs. After the enzyme is inactivated by centrifugation, the starch is treated with amylase for a period of time, and then the enzyme is inactivated, centrifuged, rotary evaporated, alcohol precipitated and dialyzed. Finally, the target product LR-CDs is obtained by drying.

[0175] Embodiment 3: According to the method described in Embodiment 2, the DP range of the LR-CDs is 9 to 50; preferably, the DP of the LR-CDs can be 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.

[0176] Embodiment 4: According to the method described in Embodiment 2 or 3, in method a or b, the mass fraction of starch in the starch suspension is 1% to 30%; in method c, the mass fraction of starch in the starch suspension is greater than 7%; preferably greater than 10%; the mass fraction can be 7% to 50%.

[0177] Embodiment 5: According to any of the methods described in Embodiments 2 to 4, the debranching enzyme may be an isoamylase, which only hydrolyzes the α-1,6 glycoside chains at the branching points of glycogen or amylopectin, cuts off the entire side branch, and forms amylose of varying lengths. The isoamylase may be a commercially available isoamylase, an isoamylase prepared by microbial fermentation, or a chemically synthesized isoamylase, and the method used may be any method known in the art; the amount of debranching enzyme added, calculated based on the substrate, is at least 100 U / g starch.

[0178] 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, Thermusancient, Thermus lettrichia, Thermus aquaticus, Streptococcus agalactiae, Corynebacterium glutamicum, Synechococcus PCC 6803, PCC 6804, Manihot esculenta, Arabidopsis thaliana, Solanum tuberosum.

[0179] The cyclodextrin glucosyltransferase may be a commercially available cyclodextrin glucosyltransferase, a cyclodextrin glucosyltransferase prepared by microbial fermentation, or a chemically synthesized cyclodextrin glucosyltransferase, and the method used may be any method known in the art;

[0180] In the cyclization reaction, the amount of cyclase added, calculated based on the substrate, can be 1 U / g starch to 20 U / g starch.

[0181] Embodiment 6: According to any of the methods described in Embodiments 2 to 5, in method a or b, the temperature for the debranching reaction can be 40°C to 80°C, and the time can be 1 to 24 hours; the temperature for the cyclization reaction can be 40°C to 80°C, and the time can be 1 to 12 hours;

[0182] In method c, the liquefaction reaction is carried out simultaneously during the gelatinization process by adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, heating it to 80-100°C and maintaining it for 20-60 minutes; the temperature for the simultaneous debranching and cyclization can be 50°C-80°C, and the time for the simultaneous debranching and cyclization is 1-48 hours.

[0183] Example 7: According to any one of the methods of claims 1 to 6, amylase is added for purification reaction; the amylase may be β-amylase or saccharifying enzyme.

[0184] Example 8: According to the method described in Example 7, the β-amylase, also known as amylo-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 a commercially available β-amylase, a β-amylase prepared by microbial fermentation, or a chemically synthesized β-amylase, and the method used can be any method known in the art.

[0185] Example 9: According to the method described in Example 7, 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. The method used can be any method known in the art.

[0186] Embodiment 10: According to any one of the methods described in Embodiments 1 to 9, 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.

[0187] Embodiment 11: According to the method described in any one of Embodiments 1 to 10, 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, 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 48 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, 19U / g starch to 20U / g starch, 21U / g starch to 22U / g starch, 22U / g starch to 23U / g starch, 23U / g starch to 24U / g starch, 24U / g starch to 25U / g starch, 25U / g starch to 26U / g starch, 26U / g starch to 27U / g starch, 27U / g starch to 28U / g starch, 28U / g starch to 29U / g starch or 29U / g starch to 30U / g starch.

[0188] Example 12: According to any one of the methods described in Examples 1 to 11, the reaction temperature of the purified amylase can be 40°C to 80°C.

[0189] Example 13: According to any one of the methods described in Examples 1 to 12, the temperature of the purification reaction can be 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, 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.

[0190] Example 14: According to any one of the methods described in Examples 1 to 13, the purification reaction time is 1 to 24 hours.

[0191] Example 15: According to any one of the methods described in Examples 1 to 14, the time for the purification reaction is: 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, 21h, 22h, 23h, 24h.

[0192] Example 16: According to any of the methods described in Examples 1 to 15, the purification step may be: alcohol precipitation, dialysis, and drying; the alcohol precipitation is: alcohol precipitation using anhydrous ethanol; the dialysis is 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 natural drying, hot air drying, vacuum drying, freeze drying, spray drying, drum drying, fluidized bed drying, infrared drying, microwave drying, and ultrasonic drying; any LR-CDs with suitable properties for use in this article 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.

[0193] Example 17: According to any of the methods described in Examples 1 to 16, the product of preparing LR-CDs from starch catalyzed by enzymes 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.

[0194] Example 18: According to the method described in any one of Examples 1 to 17, the purification and separation method is thin layer chromatography. Thin layer chromatography is a commonly used separation method, which 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.

[0195] Example 19: According to any of the methods described in Examples 1 to 18, 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 using a gel column, and the LR-CDs component is collected, and then separated using a reversed phase ODS column.

[0196] Example 20: According to the method described in any one of Examples 1 to 20, LR-CD can be purified by separation using chromatographic analysis technology. 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), CD36, CD37, CD38 and CD39 can be separated using a reverse chromatographic column and an amino column. It can also be purified by 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, 2006, 56 (1-2): 23-28). Chemistry, 2002, 44(1-4):387-390), the reaction product was eluted using an ODS chromatographic 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.

[0197] Example 21: According to any method described in Examples 1 to 20, the freeze-thaw cycle conditions are: freezing the gel sample at -80 to -20°C for 8 to 24 hours, and then thawing it in a water bath at 10 to 30°C for 1 to 5 hours, which is recorded as one freeze-thaw cycle.

[0198] Example 22: Starch gel with improved freeze-thaw stability prepared by any one of the methods described in Examples 1 to 20.

[0199] Example 23: Use of the preparation method described in any one of Examples 1 to 20 or the starch gel described in Example 23 or the LR-CDs with a degree of polymerization DP ranging from DP 10 to 50 in improving the freeze-thaw stability of frozen food.

[0200] Embodiment 24: According to the use described in Embodiment 23, the frozen food includes but is not limited to frozen pasta, frozen ready-to-eat food, frozen dessert, and frozen pet food.

[0201] The detection methods involved in the following embodiments are as follows:

[0202] Identification of LR-CDs

[0203] 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.

[0204] Example 1: Preparation and identification of LR-CDs

[0205] The specific steps are as follows:

[0206] 1. Preparation of LR-CDs

[0207] (1) Gelatinization of starch:

[0208] A certain amount of potato starch was weighed and dispersed in NaAc-HAc buffer (20 mM, pH 4.6) to prepare a 5% (w / w) potato starch solution, and heated in a boiling water bath for gelatinization for 30 min. After the solution was evenly dispersed, the starch solution was cooled to 60°C;

[0209] (2) Debranching reaction

[0210] According to the addition amount of 700U (excess) pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41, CAS: 9075-68-7, purchased from Novozymes Denmark) per gram of dry starch (based on the potato starch weighed in step (1)), pullulanase is added to the gelatinized starch solution obtained in step (1), and enzymolysis is carried out in a 60°C water bath for 24 hours. After the reaction is completed, the obtained reaction solution is boiled in a water bath for 15 minutes, centrifuged at 7000g for 20 minutes, and the supernatant is dried and then ground and sieved to obtain debranched potato starch.

[0211] (3) Secondary gelatinization reaction

[0212] Weigh a certain amount of the debranched potato starch obtained in step (2) and dissolve it in sodium citrate buffer (20 mmol / L, pH 6.0) to prepare a 2% (w / w) potato starch solution. Stir and heat in a boiling water bath for 30 min, then put it in an autoclave at 121° C. for 20 min. After gelatinization is complete, take it out and cool it down to the reaction temperature of 75° C.;

[0213] (4) Cyclization reaction

[0214] Add 10 U / g starch (based on the potato starch weighed in step (1)) of 4-α-glycosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) to the reaction system after secondary gelatinization obtained in step (3), react at 75° C. for 10 h, and terminate the reaction after boiling in a water bath for 30 min to obtain a reaction solution;

[0215] (5) Purification of LR-CDs

[0216] 30 U / g starch (based on the potato starch weighed in step (1)) of β-amylase (derived from sweet potato, EC 3.2.1.2, 974 U / mg, CAS: 9000-91-3, purchased from Sigma-Aldrich, USA) was added to the reaction solution to remove the incompletely reacted amylose. After reacting at 50° C. for 12 h, the reaction was terminated by heating in a boiling water bath for 30 min. The mixture was centrifuged at 8000 g for 30 min to remove the denatured enzyme protein precipitate. The supernatant was collected. After the supernatant was concentrated by rotary evaporation, 4 times the volume of anhydrous ethanol was added to the concentrate, and the mixture was placed at 4° C. for 12 h. After the alcohol precipitation was completed, the mixture was centrifuged at 1000 g for 10 min. The precipitate was collected, dissolved in deionized water, dialyzed against deionized water for 48 h (molecular weight cutoff 1000 Da), and freeze-dried to obtain the LR-CD sample.

[0217] 2. Identification of LR-CDs

[0218] The polymerization degree distribution of LR-CDs was determined by WATERS MALDI SYNAPT Q-TOF MS. Figure 1 As shown, LR-CDs with DP 10 to 45 were prepared.

[0219] Example 2: Effect of LR-CDs on freeze-thaw stability of potato starch

[0220] The specific steps are as follows:

[0221] (1) Accurately weigh 2 g of potato starch, add water to prepare a starch suspension with a potato starch mass fraction of about 7%, add 5% (starch dry basis percentage, w / w) of LR-CDs with DP 10-45 prepared in Example 1, and stir until completely dissolved to obtain a sample;

[0222] (2) The sample, after being stirred until completely dissolved, was placed in a rapid viscosity analyzer (RVA) and gelatinized according to the set program. The specific procedure was as follows: the sample was kept at 50°C for 1 min, then heated to 95°C at a heating rate of 12.2°C / min and kept at 95°C for 2.5 min; then the sample was cooled to 50°C at a rate of 11.8°C / min and kept at this temperature for 2 min.

[0223] All the above steps require stirring. The speed of the stirring paddle is 960 r / min within 10 s at the beginning of the experiment to make the sample evenly dispersed, and then the speed is fixed at 160 r / min.

[0224] (3) After gelatinization, the sample was collected into a 50 mL centrifuge tube and refrigerated at 4°C for 24 h to form a gel. The obtained gel sample was used to determine the freeze-thaw stability.

[0225] (4) The freeze-thaw stability of starch gel is usually measured by the dehydration rate during the freeze-thaw cycle. The gel sample is frozen at -20°C for 12 hours, and then thawed in a 25°C water bath for 2 hours. This is recorded as one freeze-thaw cycle. The dehydration rate after each thawing is measured: the thawed starch gel is centrifuged at 4000r / min for 15 minutes, the supernatant is discarded, the centrifuge tube is tilted 45°, and after standing for 10 minutes, the supernatant is discarded again, and the water attached to the tube wall is absorbed and then weighed. The dehydration rate is calculated according to formula (1):

[0226] Separation rate = (M1-M2) / M(1)

[0227] Where: M is the mass of the sample (g); M1 is the mass of the sample and the centrifuge tube before centrifugation (g); M2 is the mass of the sample and the centrifuge tube after centrifugation (g).

[0228] When starch gel is frozen, most of the water is frozen into ice crystals, and starch and a small portion of unfrozen water form starch-rich areas. During the thawing process, the ice crystals melt into water, and the water is released from the gel structure. Repeated freezing and thawing accelerates the formation of larger ice crystals and the combination of starch chains, resulting in a gradual increase in the water extraction rate in the gel system. Therefore, the ability of starch to resist adverse physical changes during freezing and thawing is often referred to as freeze-thaw stability, which can be evaluated by the water extraction rate index.

[0229] Potato starch (weighing 2 g of potato starch, adding water to prepare a starch suspension with a potato starch mass fraction of about 7%) was used as a control and treated in the same manner as above, except that LR-CDs (DP 10-45) was not added, and the rest of the operations remained unchanged.

[0230] The results are shown in Table 1.

[0231] Table 1 Effect of freeze-thaw cycles on the water separation rate of potato starch gel

[0232]

[0233] As shown in Table 1, during the five freeze-thaw cycles, the water separation rate of the starch gel with the addition of LR-CDs (DP 10-45) was lower than that of the original starch gel, which indicates that LR-CDs (DP 10-45) has a good inhibitory effect on the dehydration of starch gel during the freeze-thaw cycle, and the addition of LR-CDs (DP 10-45) significantly improves the freeze-thaw stability of the gel.

[0234] Example 3: Preparation of LR-CDs-1 with a degree of polymerization ranging from 9 to 36

[0235] (1) Preparation of high concentration starch:

[0236] 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).

[0237] (2) Gelatinization and liquefaction of high-concentration starch:

[0238] 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;

[0239] 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. The enzyme was then inactivated for 30 minutes.

[0240] The DE of starch after gelatinization and liquefaction reaction is: 2~8.

[0241] (3) Synchronous action of pullulanase debranching and 4-α-glycosyltransferase cyclization:

[0242] 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.

[0243] (4) β-amylase treatment

[0244] 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.

[0245] (5) Alcohol precipitation treatment

[0246] 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.

[0247] (6) Removal of small molecule sugars by dialysis:

[0248] 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.

[0249] (7) Freeze drying:

[0250] 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.

[0251] The method of this embodiment can prepare LR-CDs-1 with a degree of polymerization ranging from 9 to 36.

[0252] Example 4: Effect of LR-CDs-1 on freeze-thaw stability of potato starch

[0253] The specific implementation is the same as Example 2, except that LR-CDs is replaced by LR-CDs-1 in Example 3, and the specific operation remains unchanged.

[0254] The results are shown in Table 2.

[0255] Table 2 Effect of freeze-thaw cycles on the water separation rate of potato starch gel

[0256]

[0257] As shown in Table 2, during the five freeze-thaw cycles, the water separation rate of the starch gel with the addition of LR-CDs-1 (DP9-36) was lower than that of the original starch gel. This indicates that LR-CDs-1 has a good inhibitory effect on the dehydration of starch gel during the freeze-thaw cycle, and the addition of LR-CDs-1 significantly improves the freeze-thaw stability of the gel.

[0258] Therefore, LR-CDs with different DP ranges also have a gel-enhancing effect on cassava starch gel.

[0259] Example 5: Effect of LR-CDs on the gel texture of potato starch

[0260] The specific steps are as follows:

[0261] (1) The starch paste after RVA gelatinization in Example 2 (obtained in step (2)) was collected into a disposable plastic culture dish with a diameter of 36 mm and a height of 1 mm and covered with plastic wrap, cooled to room temperature (25° C.), and refrigerated at 4° C. for 24 h to form a gel;

[0262] (2) Place the gel sample evenly on the TA-XT plus texture analyzer platform. Test conditions: Probe model: P36R; Measurement height: 15 mm; Pre-test speed: 0.5 mm / s; Test speed: 0.5 mm / s; Post-test speed: 0.5 mm / s; Trigger force 5 g, deformation amount 50%.

[0263] Potato starch (weighing 2 g of potato starch, adding water to prepare a starch suspension with a potato starch mass fraction of about 7%) was used as a control and treated in the same manner as above, except that LR-CDs (DP 10-45) was not added, and the rest of the operations remained unchanged.

[0264] The results are shown in Table 3.

[0265] Table 3 Changes in potato starch gel texture parameters after adding LR-CDs (DP 10-45)

[0266]

[0267] It can be seen from Table 3 that the potato starch gel formed by adding LR-CDs (DP 10-45) has a reduced hardness, a slightly increased elasticity, and no significant differences in cohesion and recovery.

[0268] Example 6: Effect of LR-CDs (DP 10-45) on freeze-thaw stability of wheat starch

[0269] The potato starch in Example 2 was replaced with wheat starch, and the other operations were the same. Wheat starch gel was prepared according to the method of Examples 1 and 2; the effect of adding LR-CDs (DP 10-45) on the freeze-thaw stability of wheat starch was studied. The freeze-thaw stability detection method was the same as that of Example 2.

[0270] Meanwhile, wheat starch (weighing 2 g of wheat starch, adding water to prepare a starch suspension with a wheat starch mass fraction of about 7%) was used as a control and treated in the same manner as above, except that LR-CDs (DP 10-45) were not added, and the rest of the operations remained unchanged.

[0271] The results are shown in Table 4.

[0272] Table 4 Effect of freeze-thaw cycles on the water separation rate of wheat starch gel

[0273]

[0274] The results showed that in the first two freeze-thaw cycles, no water was released from the wheat starch gel with the addition of LR-CDs (DP 10-45), and LR-CDs (DP 10-45) had a significant inhibitory effect on the water release of wheat starch gel. After three freeze-thaw cycles, the water release rate of the gel increased significantly. After five freeze-thaw processes, the water release rate of the wheat starch gel with the addition of LR-CDs (DP 10-45) was lower than that of the original starch gel. The addition of LR-CDs (DP 10-45) also improved the freeze-thaw stability of the wheat starch gel.

[0275] Example 7: SEM microstructure observation of starch gel of LR-CDs

[0276] Specific implementation method The same as Examples 1-2, potato starch gel was prepared according to the method of Example 2; the SEM microstructure of the starch gel with LR-CDs (DP 10-45) added was detected, and the specific steps were as follows:

[0277] (1) The starch paste after RVA gelatinization in Example 2 (obtained in step (2)) was collected in a 50 mL centrifuge tube and placed in a 4° C. refrigerator for 24 h. After the sample formed a gel, the sample was placed in liquid nitrogen for 5 min. The liquid nitrogen quenching reduced the growth of ice crystals and caused the water in the sample to freeze rapidly in situ.

[0278] (2) The frozen sample is then placed in a freeze dryer for freeze drying. The frozen part inside the sample is cut with a blade and fixed on the conductive glue on the sample stage. After gold spraying and vacuuming, the sample is scanned under an accelerating voltage to observe and photograph the representative part.

[0279] Potato starch (weighing 2 g of potato starch, adding water to prepare a starch suspension with a potato starch mass fraction of about 7%) was used as a control and treated in the same manner as above, except that LR-CDs (DP 10-45) was not added, and the rest of the operations remained unchanged.

[0280] The results are as follows Figure 2 As shown in the figure, the gel formed by starch gelatinization fully extends during the cooling process, and the molecules reaggregate to form a honeycomb-like network structure. The wall layer of the network structure in the original starch gel is thin and rough, which may be due to the loss of water during the freeze-drying process, resulting in damage to the network structure. The addition of LR-CDs (DP 10-45) improves the network structure of the gel, making it have a more uniform and dense network structure, the pores of the system are reduced, and its wall layer becomes flatter and smoother. This may be because LR-CDs (DP 10-45) contain a large number of hydrophilic group hydroxyls, which improves the water holding capacity of the gel. This is consistent with the result that LR-CDs (DP 10-45) improves the freeze-thaw stability of starch gel.

[0281] Example 8: Effect of LR-CDs prepared by different debranching enzymes on the freeze-thaw stability of potato starch gel

[0282] The specific steps are as follows:

[0283] The specific implementation method for preparing LR-CDs is the same as that in Example 1, except that the pullulanase in the debranching reaction of step (2) is replaced with isoamylase (Aspergillus niger, EC 3.2.1.57, 1000 U / mL, purchased from Megazyme Ireland) to debranch the starch substrate, and the other operations remain unchanged. After LC-MS identification, LR-CDs with DP 16-42 were prepared.

[0284] Referring to the method of Example 2, 5% (starch dry basis percentage) LR-CDs (DP 16-42) was added to 7% (w / w) potato starch to participate in gelatinization, and the gel was subjected to 5 freeze-thaw cycles to determine its water separation rate. Potato starch (weighing 2g potato starch, adding water to prepare a starch suspension with a potato starch mass fraction of about 7%) was used as a control and treated in the same manner according to the above steps, except that LR-CDs (DP 16-42) was not added, and the other operations remained unchanged. The results are shown in Table 5.

[0285] Table 5 Effect of different debranching enzymes on the gel separation rate of potato starch after LR-CDs (DP 16-42) were prepared

[0286]

[0287] As shown in Table 5, during the five freeze-thaw cycles, the water separation rate of the starch gel with the addition of LR-CDs (DP 16-42) was lower than that of the original starch gel. This indicates that the LR-CDs (DP 16-42) prepared by debranching the starch substrate with different debranching enzymes can still have a good inhibitory effect on the dehydration of the starch gel during the freeze-thaw cycle, and the addition of LR-CDs improves the freeze-thaw stability of the gel.

[0288] Example 9: Effect of LR-CDs prepared by different glycosyltransferases on freeze-thaw stability of potato starch gel

[0289] The specific implementation method for preparing LR-CDs is the same as Example 1, except that the 4-α-glycosyltransferase from Thermoproteus uzoniensis (NCBI accession number: WP_013679179.1) is replaced with 4-α-glycosyltransferase from other sources shown in Table 6, and the other conditions remain unchanged.

[0290] Referring to the method of Example 2, LR-CDs with different DP ranges prepared by enzymes from different sources were added to 7% (w / w) potato starch at an addition amount of 5% (w / w, starch dry basis percentage) to participate in gelatinization, and the gel was subjected to 5 freeze-thaw cycles to measure its water separation rate. The results are shown in Table 6.

[0291] Table 6 Effect of different glycosyltransferases on the gelation rate of potato starch after LR-CDs were prepared

[0292]

[0293] As shown in Table 6, during the five freeze-thaw cycles, the dehydration rate of the starch gel with added LR-CDs was lower than that of the original starch gel. This indicates that the LR-CDs with different DP prepared by 4-α-glycosyltransferases from different sources can still have a good inhibitory effect on the dehydration of starch gel during the freeze-thaw cycle, thereby improving the freeze-thaw stability of the gel.

[0294] Example 10: Effects of adding different LR-CDs and adjusting starch concentration on freeze-thaw stability of gel

[0295] Referring to the method of Example 2, LR-CDs with different addition amounts (w / w, starch dry basis percentage) were added to potato starch with different concentrations (w / w) to participate in gelatinization, and the subsequent gel was subjected to 5 freeze-thaw cycles to measure its water separation rate. The results are shown in Table 7.

[0296] Table 7 Effects of adding different LR-CDs and adjusting starch concentration on water separation rate

[0297]

[0298] As shown in Table 7, during the five freeze-thaw cycles, the dehydration rate of the starch gel with LR-CDs added was lower than that of the original starch gel. The gel with a higher starch concentration had a lower dehydration rate, and for the gel with the same starch concentration, the dehydration rate would not decrease further if the amount of LR-CDs added was increased.

[0299] In summary, the results show that different addition amounts of LR-CDs can inhibit gel dehydration during freeze-thaw cycles of gels with different starch concentrations (5% to 20%) and can improve the freeze-thaw stability of the gel.

[0300] 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 improving the freeze-thaw stability of starch gel, characterized in that: The method comprises the following steps: dispersing starch in water to prepare a starch suspension, adding macrocyclodextrin (LR-CDs) having a degree of polymerization (DP) of 9 to 50 to the starch suspension, and gelatinizing the suspension to obtain a starch gel; preferably, the DP of the LR-CDs may be 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, 20 to 21, 21 to 22. , 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 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.

2. The method according to claim 1, characterized in that The mass fraction of starch in the starch suspension is 5-30% (w / w); preferably, the mass fraction of starch in the starch suspension is 5%-6% (w / w), 6%-7% (w / w), 7%-8% (w / w), 8%-9% (w / w), 9%-10% (w / w), 10%-11% (w / w), 11%-12% (w / w), 12%-13% (w / w), 13%-14% (w / w), 14%-15% (w / w), 15%-16% (w / w), 16% ~ 17% (w / w), 17% ~ 18% (w / w), 18% ~ 19% (w / w), 19% ~ 20% (w / w), 21% ~ 22% (w / w), 22% ~ 23% (w / w), 23% ~24% (w / w), 24% ~ 25% (w / w), 25% ~ 26% (w / w), 26% ~ 27% (w / w), 27% ~ 28% (w / w), 28% ~ 29% (w / w) or 29% ~ 30% (w / w); Preferably, the amount of the macrocyclodextrin (LR-CDs) added is 1% to 20% (w / w) of the dry weight of starch, calculated on the basis of the amount of starch added; Preferably, the addition amount of the macrocyclodextrin (LR-CDs), calculated as the addition amount of starch, is 1% to 2% (w / w), 2% to 3% (w / w), 3% to 4% (w / w), 4% to 5% (w / w), 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w), 15% to 16% (w / w), 16% to 17% (w / w), 17% to 18% (w / w), 18% to 19% (w / w) or 19% to 20% (w / w) of the dry weight of starch.

3. The method according to claim 1 or 2, characterized in that: The gelatinization conditions are: heating at 90-100° C. for 30-60 minutes to completely gelatinize the starch.

4. The method according to any one of claims 1 to 3, characterized in that: The dehydration rate of starch gel decreases during several freeze-thaw cycles, and the freeze-thaw stability is improved; the conditions of the freeze-thaw cycle are: the gel sample is frozen at -80 to -20°C for 8 to 24 hours, and then thawed in a water bath at 10 to 30°C for 1 to 5 hours, which is recorded as one freeze-thaw cycle.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The preparation method of LR-CDs comprises the following steps: gelatinizing starch and then adding a debranching enzyme to perform debranching, adding a glycosyltransferase to perform cyclization after debranching, and adding an amylase to perform purification after cyclization to prepare LR-CDs; Preferably, the debranching enzyme includes but is not limited to: pullulanase, isoamylase; Preferably, 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; Preferably, the glycosyltransferase includes but is not limited to: 4-α-glycosyltransferase, cyclodextrin glucosyltransferase; Preferably, the cyclization conditions are: adding 1 to 100 U / g starch glycosyltransferase, and enzymolysis at 40 to 80° C. for 1 to 12 hours; Preferably, the amylase includes but is not limited to: β-amylase, saccharifying enzyme; Preferably, the conditions for the amylase treatment are: adding 1 to 100 U / g of starch amylase and performing enzymolysis at 40 to 80° C. for 1 to 24 hours; Preferably, the purification step is to prepare LR-CDs after amylase treatment, centrifugation, rotary evaporation, alcohol precipitation, centrifugation and dialysis.

6. The preparation method according to any one of claims 1 to 4, characterized in that: The preparation method of the LR-CDs 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 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 react and prepare LR-CDs; Preferably, the mass fraction of starch in the starch suspension is 7% to 50%; Preferably, 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 basis of substrate, 1U / g starch to 20U / g starch; the addition amount of the liquefaction enzyme is: calculated on the basis of substrate, 1U / g starch to 20U / g starch; Preferably, 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 it to 80-100° C., and maintaining it 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 20U / g starch; Preferably, the liquefying enzyme is 4-α-glycosyltransferase; 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; the amount of the debranching enzyme added is, based on the substrate, at least 100U / g starch of the debranching enzyme is added; the amount of the cyclase added is: based on the substrate, 1U / g starch to 100U / g starch; Preferably, in step (2), the reaction conditions are: reaction at 50°C to 80°C for 1 to 48 hours; Preferably, 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.

7. The preparation method according to claim 6, characterized in that: The starch includes but is not limited to potato starch, corn starch, cassava starch, and pea starch; preferably, the mass fraction of starch in the starch suspension is 1% to 2% (w / w), 2% to 3% (w / w), 3% to 4% (w / w), 4% to 5% (w / w), 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w). / w), 14% ~ 15% (w / w), 15% ~ 16% (w / w), 16% ~ 17% (w / w), 17% ~ 18% (w / w), 18% ~ 19% (w / w), 19% ~ 20% (w / w), 21% ~ 22% (w / w), 22% ~ 23% (w / w), 23% to 24% (w / w), 24% to 25% (w / w), 25% to 26% (w / w), 26% to 27% (w / w), 27% to 28% (w / w), 28% to 29% (w / w) or 29% to 30% (w / w).

8. Starch gel with improved freeze-thaw stability prepared by the method according to any one of claims 1 to 7.

9. Use of the preparation method according to any one of claims 1 to 7 or the starch gel according to claim 8 or the LR-CDs with a degree of polymerization DP ranging from DP 9 to 50 in improving the freeze-thaw stability of frozen food.

10. The use according to claim 9, characterized in that: The frozen food includes but is not limited to frozen pasta, frozen ready-to-eat food, frozen dessert, and frozen pet food.

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