Method for regulating and controlling starch gel strength

Abstract

The invention discloses a method for regulating and controlling starch gel strength, and belongs to the technical field of starch processing. Macrocyclodextrin LR-CDs is prepared through 4-alpha-glycosyl transferase, the macrocyclodextrin LR-CDs is added into potato starch, cassava starch, corn starch, wheat starch and pea starch to participate in the gelatinization process together, the adding amount is 1%-10% of the dry basis weight of the starch, and starch gel is formed after gelatinization and cooling. The hardness of the starch gel can be in a change trend of first increasing and then decreasing along with the increase of the addition amount according to the difference of the addition amount of the LR-CDs, so that the strength of the starch gel is regulated and controlled. The method belongs to physical modification by adding homologous substances, is green and environment-friendly, and does not bring potential safety hazards. The structural strength and functional characteristics of the gel material can be balanced by precisely regulating and controlling the gel strength, and the method is a key for realizing multi-scene adaptive application of the starch-based material and has important significance on application of the starch-based material in food processing.

Description

Technical Field

[0001] The present invention relates to a method for regulating the gel strength of starch, which is used to improve the disadvantages of too soft or too hard starch gel and poor stability, and belongs to the technical field of starch processing. Background Art

[0002] Starch is a natural polymer carbohydrate that is insoluble in cold water. However, when heated to a certain temperature, starch granules can absorb water and swell, and water molecules enter the interior of the starch granules and combine with some starch molecules in the form of hydrogen bonds. First, the amorphous region of starch is destroyed, and then the crystalline region of starch is affected, causing the internal structure of starch molecules to be disrupted. Macroscopically, starch gradually forms a semi-transparent viscous liquid from a suspension, and this process is called the gelatinization of starch. During the cooling and standing process of gelatinized starch, the linear amylose molecular chains crosslink and polymerize through hydrogen bonds to form a continuous three-dimensional network gel structure. The swollen starch granules and fragments are filled in the amylose network to form a gel with a certain strength and viscoelasticity.

[0003] Starch gel has biocompatibility, non-toxicity, and biodegradability, and is widely used in the food, pharmaceutical, and cosmetic industries. However, during the food production process, natural starch gel has problems such as easy breakage or poor texture properties (such as elasticity, chewiness, etc.). In order to maintain the integrity of the product structure, foods based on starch gel (such as processed meat, jellied bean curd, jelly, vermicelli, etc.) need to have a certain gel strength to resist severe deformation or even breakage.

[0004] Natural cassava starch, like corn starch and potato starch, is widely used as a processing raw material or ingredient in China's food industry. Currently, the apparent domestic demand for cassava starch also exceeds 3 million tons per year. The amylose content in cassava starch is lower than that of other starches containing amylose, and the molecular weights of amylose and amylopectin are high. The specific structure results in the unique gel properties of this type of starch. The gel of cassava starch is relatively soft and has poor stability. This limits its application in the production of various foods.

[0005] In the food industry, chemical modification methods including cross-linking modification are often used to improve the gelatinization and gelation properties of cassava starch. Cross-linking modification introduces covalent linkages between starch molecules, enhancing the acid resistance, high-temperature resistance, shear resistance, etc. of starch; in addition, moderate cross-linking modification can strengthen the starch granule structure, making it not easy to disintegrate during the gelatinization process, increasing the final viscosity of starch gelatinization, and enabling the swollen starch granules to better embed in the amylose network structure, thereby enhancing the gel strength of starch. Although cross-linked starch has good application performance in many aspects, low-degree cross-linking modification cannot meet the requirement of cassava gel strength, while high-degree cross-linked cassava starch will deteriorate the taste, and there are also safety hazards brought by chemical modification. Therefore, it is of great significance to improve the gel strength of cassava starch while ensuring the sensory quality of the sample, for improving the performance of cassava starch-based foods and expanding the application of cassava starch in the food field.

[0006] Too low or too high starch gel strength will limit its application value. When the gel strength is insufficient, the network structure is loose and easy to collapse, resulting in problems such as easy breakage of noodles in the food industry, serious ice crystal puncture of frozen foods, increased risk of burst release of drug delivery systems in the pharmaceutical field, and decreased bonding strength of industrial colloid materials; at the same time, the weakened water-holding capacity is prone to cause gel syneresis, affecting the texture and shelf life of products. While too high gel strength will form a dense and rigid network, causing problems such as a stiff taste of food, poor swallowing palatability, hindered drug diffusion in sustained-release preparations, and difficult extrusion molding of 3D printing materials. At the same time, excessive cross-linking will also increase processing energy consumption and reduce the bioavailability of nutritional components. Precise regulation of gel strength can balance the structural strength and functional characteristics of materials, and is the key to realizing the multi-scenario adaptable application of starch-based materials.

[0007] The existing methods for regulating starch gel strength are mainly based on physical, chemical, and biological modification means. In terms of physical modification, heat-moisture treatment, high-pressure homogenization, or ultrasonic treatment are often used to change the morphology and crystal structure of starch granules, and the gel network density is affected by regulating the degree of gelatinization; chemical modification mainly includes cross-linking, esterification, or etherification, which enhances the intermolecular force by introducing covalent bonds, but may introduce chemical residues; biological modification uses starch branching enzyme or pullulanase, etc. to directionally cut or recombine glycosidic bonds to optimize the molecular chain distribution. In addition, compound plasticizers (polyols), colloids (xanthan gum), or nanofillers (cellulose nanocrystals) can strengthen the gel network through filling effects or interfacial actions. Summary of the Invention

[0008] The inventors prepared LR-CDs and added them to cassava starch to participate in the gelatinization process together. After gelatinization and cooling, the gel strength of the formed starch gel was enhanced. The preparation process of the present invention is efficient, environmentally friendly, has simple equipment, and good product effects. This gel is suitable for producing puddings, fillings, improving the quality of meat products, etc., and has a very wide application in the food field. In this article, unless otherwise specified, (w / v) is g / mL (mass / volume), and (w / w) is the mass fraction.

[0009] The present invention provides an application of LR-CDs with a DP greater than 8 in increasing the gel strength of starch.

[0010] In one embodiment of the present invention, the DP range of the LR-CDs is 9 to 50, and the degree of polymerization of the LR-CDs includes DP X, where X is any value from 9 to 50, such as DP 9, DP 10, etc.

[0011] In one embodiment of the present invention, the DP of the LR-CDs includes A to B, where A is any value from 9 to 49, B is greater than A, and B is any value from 10 to 50. The DP of the LR-CDs can 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 to 23, 23 to 24, 24 to 25, 25 to 26, 26 to 27, 27 to 28, 28 to 29, 29 to 30, 30 to 31, 31 to 32, 32 to 33, 33 to 34, 34 to 35, 35 to 36, 36 to 37, 37 to 38, 38 to 39, 39 to 40, 40 to 41, 41 to 42, 42 to 43, 43 to 44, 44 to 45, 45 to 46, 46 to 47, 47 to 48, 48 to 49 or 49 to 50.

[0012] In one embodiment of the present invention, the application is to disperse starch in water to form a starch suspension, and add the LR-CDs to the starch suspension to participate in gelatinization together, and then obtain a starch gel with enhanced gel strength.

[0013] In one embodiment of the present invention, the mass fraction of starch in the starch suspension is 5-30% (w / w). Optionally, 5-20% (w / w); optionally, 5-15% (w / w); optionally, 5-10% (w / w); optionally, 6% (w / w); further optionally, 7% (w / w); optionally, 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).

[0014] In one embodiment of the present invention, the addition amount of the LR-CDs, based on the addition amount of starch, is 1%-10% (w / w) of the dry weight of starch;

[0015] In one embodiment of the present invention, the addition amount of the macrocyclic dextrin (LR-CDs), based on the addition amount of starch, 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) of the dry weight of starch.

[0016] In one embodiment of the present invention, the gelatinization conditions are: 90-100 °C, heating for 30-60 min to completely gelatinize the starch.

[0017] In one embodiment of the present invention, the gelatinization includes, but is not limited to, RVA instrument gelatinization and water bath pot gelatinization.

[0018] In one embodiment of the present invention, the specific steps of the gelatinization are as follows:

[0019] After adding LR-CDs to the starch suspension, magnetic stirring is carried out for 1-10 min to disperse the starch particles evenly. It is placed in a water bath pot with magnetism, and magnetic stirring is carried out at 80-100 °C for 5-30 min to completely gelatinize it.

[0020] In one embodiment of the present invention, the preparation method of the LR-CDs is as follows: using starch as a raw material, after first subjecting it to debranching treatment with debranching enzyme, adding an appropriate amount of glycosyltransferase, selecting a suitable addition amount and action time to produce LR-CDs. After inactivating the enzyme and centrifuging, and then treating with amylase for a period of time, inactivating the enzyme, centrifuging, rotary evaporation, alcohol precipitation and dialysis are carried out, and finally drying to obtain the target product LR-CDs. Adding the prepared LR-CDs to starch and gelatinizing them together can quickly obtain a cassava starch gel with increased gel strength.

[0021] In one embodiment of the present invention, the debranching enzyme includes, but is not limited to: pullulanase (EC 3.2.1.41), isoamylase (EC 3.2.1.68).

[0022] In one embodiment of the present invention, the debranching conditions are as follows: adding 10 - 1000 U / g starch of debranching enzyme and enzymatically hydrolyzing at 40 - 80 °C for 1 - 24 h;

[0023] In one embodiment of the present invention, the glycosyltransferase includes, but is not limited to: 4-α-glycosyltransferase (EC2.4.1.25), cyclodextrin glucosyltransferase (EC 2.4.1.19).

[0024] In one embodiment of the present invention, the NCBI accession numbers of the 4-α-glycosyltransferase include, but are not limited to: WP_013679179.1, ABP49821.1, AFA40300.1, AET33957.1, ESQ23201.1, ESQ23782.1.

[0025] In one embodiment of the present invention, the cyclization conditions are as follows: adding 1 - 100 U / g starch of glycosyltransferase and enzymatically hydrolyzing at 40 - 80 °C for 1 - 12 h;

[0026] In one embodiment of the present invention, the amylase includes, but is not limited to: β-amylase (EC 3.2.1.2), glucoamylase (EC 3.2.1.3).

[0027] In one embodiment of the present invention, the conditions for amylase treatment are as follows: adding 1 - 100 U / g starch of amylase and enzymatically hydrolyzing at 40 - 80 °C for 1 - 24 h;

[0028] In one embodiment of the present invention, the purification step is to prepare LR-CDs after treatment with amylase, followed by centrifugation, rotary evaporation, alcohol precipitation, centrifugation and dialysis;

[0029] In one embodiment of the present invention, ethanol is used for alcohol precipitation, and the addition ratio of the reaction solution to ethanol is 1:1 to 1:10.

[0030] In one embodiment of the present invention, the cut-off molecular weight of the dialysis bag is 500 - 3000 Da.

[0031] In one embodiment of the present invention, the drying method includes freeze drying, atmospheric drying, spray drying, drum drying or microwave drying.

[0032] In one embodiment of the present invention, the starch includes but is not limited to potato starch, corn starch, cassava starch, and pea starch.

[0033] The present invention also provides another method for preparing LR-CDs, and the method includes the following steps:

[0034] (1) Adding a liquefying enzyme to the starch suspension, and synchronously performing 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 further preferably greater than 20%;

[0035] (2) Adding a debranching enzyme and a cyclizing enzyme to the starch obtained in step (1) simultaneously, and reacting to obtain a reaction product; or adding a debranching enzyme to the starch obtained in step (1) first for a debranching reaction, and then adding a cyclizing enzyme for reaction to obtain a reaction product;

[0036] (3) Adding an amylase to the reaction product obtained in step (2) for reaction to prepare LR-CDs;

[0037] In one embodiment of the present invention, in step (1), the liquefying enzyme includes but is not limited to: α-amylase, 4-α-glucosyltransferase, cyclodextrin glucosyltransferase; when the liquefying enzyme is 4-α-glucosyltransferase, the addition amount is: calculated based on the substrate, 1 U / g starch - 20 U / g starch; when the liquefying enzyme is α-amylase, the addition amount is: calculated based on the substrate, 1 U / g starch - 20 U / g starch; when the liquefying enzyme is cyclodextrin glucosyltransferase, the addition amount is: calculated based on the substrate, 1 U / g starch - 20 U / g starch.

[0038] In one embodiment of the present invention, in step (1), the synchronous liquefaction reaction during the gelatinization process means that after adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, the temperature is raised to 80 - 100 °C and maintained for 20 - 60 min;

[0039] In one embodiment of the present invention, after the synchronous liquefaction reaction during the gelatinization process, the DE value of the starch is controlled to be 2 - 8.

[0040] In one embodiment of the present invention, the addition amount of the liquefying enzyme is: 1 U / g starch to 20 U / g starch;

[0041] In one embodiment of the present invention, the liquefying enzyme is 4-α-glucosyltransferase; the debranching enzyme includes but is not limited to: pullulanase, isoamylase, amylopectinase; the cyclizing enzyme includes but is not limited to: 4-α-glucosyltransferase, cyclodextrin glucosyltransferase; the addition amount of the debranching enzyme is, based on the substrate, adding at least 100 U / g starch of the debranching enzyme; the addition amount of the cyclizing enzyme is: calculated based on the substrate, 1 U / g starch to 20 U / g starch;

[0042] In one embodiment of the present invention, in step (2), the reaction conditions are: reacting at 50°C to 80°C for 1 to 48 hours;

[0043] In one embodiment of the present invention, in step (3), the amylase is β-amylase, glucoamylase; the addition amount of the amylase is, calculated based on the substrate, adding 1 U / g starch to 100 U / g starch; the reaction temperature after adding the amylase is 30°C to 60°C, and the reaction time is 1 to 48 hours.

[0044] In one embodiment of the present invention, the mass fraction of starch in the starch suspension is 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w), 15% to 16% (w / w), 16% to 17% (w / w), 17% to 18% (w / w), 18% to 19% (w / w), 19% to 20% (w / w), 21% to 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);

[0045] In one embodiment of the present invention, the addition amount of the macrocyclic dextrin (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) of the dry weight of the starch.

[0046] The present invention also provides an application of macrocyclic dextrins (LR-CDs) with a degree of polymerization (DP) greater than 8 in regulating the gel strength of starch.

[0047] In one embodiment of the present invention, the application is as follows: starch is dispersed in water to form a starch suspension, and the LR-CDs are added to the starch suspension for gelatinization. After that, with different addition amounts, starch gels with enhanced or weakened gel strength are obtained.

[0048] In one embodiment of the present invention, 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, 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.

[0049] In one embodiment of the present invention, the application is as follows: starch is dispersed in water to form a starch suspension, and the LR-CDs are added to the starch suspension to participate in gelatinization together. After that, starch gels with enhanced or weakened gel strength are obtained.

[0050] In one embodiment of the present invention, the mass fraction of starch in the starch suspension is 5 - 50%.

[0051] In one embodiment of the present invention, 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), 20% - 21% (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), 29% - 30% (w / w), 30% - 31% (w / w), 31% - 32% (w / w), 32% - 33% (w / w), 33% - 34% (w / w), 34% - 35% (w / w), 35% - 36% (w / w), 36% - 37% (w / w), 37% - 38% (w / w), 38% - 39% (w / w), 39% - 40% (w / w), 40% - 41% (w / w), 41% - 42% (w / w), 42% - 43% (w / w), 43% - 44% (w / w), 44% - 45% (w / w), 45% - 46% (w / w), 46% - 47% (w / w), 47% - 48% (w / w), 48% - 49% (w / w) or 49% - 50% (w / w);

[0052] In one embodiment of the present invention, the addition amount of the LR-CDs, based on the addition amount of starch, is 1% - 10% (w / w) of the dry basis weight of starch;

[0053] In one embodiment of the present invention, the addition amount of the macrocyclic dextrin (LR-CDs), based on the addition amount of starch, 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) or 9% - 10% (w / w) of the dry basis weight of starch.

[0054] In one embodiment of the present invention, the gelatinization conditions are: 90 - 100 °C, heating for 30 - 60 min to completely gelatinize the starch.

[0055] The present invention provides a method for increasing the gel strength of cassava starch. The method is to disperse cassava starch in water to prepare a starch suspension, and add LR-CDs with a degree of polymerization DP in the range of 9-50 to the starch suspension for gelatinization to obtain a cassava starch gel with enhanced gel strength.

[0056] In one embodiment of the present invention, the mass fraction (w / w) of cassava starch in the starch suspension is 5-30%;

[0057] In one embodiment of the present invention, the addition amount of the LR-CDs, based on the addition amount of cassava starch, is 1%-10% (w / w) of the dry weight of cassava starch.

[0058] In one embodiment of the present invention, the conditions for gelatinization are: 90-100 °C, heating for 30-60 min to completely gelatinize the starch.

[0059] In one embodiment of the present invention, the gelatinization includes, but is not limited to, RVA instrument gelatinization and water bath pot gelatinization;

[0060] In one embodiment of the present invention, the gelatinization specifically includes the following steps:

[0061] (1) After adding LR-CDs to the starch suspension, keep it at 45-55 °C for 1-5 min, then increase the temperature at a rate of 10-15 °C / min to 90-100 °C, and keep it at 90-100 °C for 1-5 min;

[0062] (2) Cool the sample obtained in step (1) to 45-55 °C at a rate of 10-15 °C / min and keep it at 45-55 °C for 1-5 min; The entire gelatinization process needs to be stirred. Within 1-30 s from the start of gelatinization, the stirring paddle stirs at a speed of 500-1000 r / min, and after 1-30 s of the gelatinization process, adjust the rotation speed to 100-200 r / min.

[0063] In one embodiment of the present invention, the gelatinization specifically includes the following steps:

[0064] After adding LR-CDs to the starch suspension, stir magnetically for 1-10 min to disperse the starch particles evenly. Place it in a water bath pot with magnetic force, and stir magnetically at 80-100 °C for 5-30 min to completely gelatinize it.

[0065] In one embodiment of the present invention, the preparation method of the LR-CDs is as follows: using starch as a raw material, after debranching treatment with debranching enzyme, an appropriate amount of glycosyltransferase is added, and the appropriate addition amount and action time are selected to produce LR-CDs. After inactivating the enzyme and centrifuging, it is treated with amylase for a period of time, then the enzyme is inactivated, centrifuged, rotary evaporated, ethanol precipitated and dialyzed, and finally dried to obtain the target product LR-CDs.

[0066] In one embodiment of the present invention, the debranching enzyme includes, but is not limited to: pullulanase (EC 3.2.1.41), isoamylase (EC 3.2.1.57).

[0067] In one embodiment of the present invention, the debranching conditions are as follows: adding 10 - 1000 U / g of starch debranching enzyme, and enzymatically hydrolyzing at 40 - 80 °C for 1 - 24 h;

[0068] In one embodiment of the present invention, the glycosyltransferase includes, but is not limited to, those derived from: 4-α-glycosyltransferase (EC 2.4.1.25), cyclodextrin glucosyltransferase (EC 2.4.1.19).

[0069] In one embodiment of the present invention, the cyclization conditions are as follows: adding 1 - 100 U / g of starch glycosyltransferase, and enzymatically hydrolyzing at 40 - 80 °C for 1 - 12 h;

[0070] In one embodiment of the present invention, the amylase includes, but is not limited to: β-amylase (EC 3.2.1.2), glucoamylase (EC 3.2.1.3).

[0071] In one embodiment of the present invention, the conditions for amylase treatment are as follows: adding 1 - 100 U / g of starch amylase, and enzymatically hydrolyzing at 40 - 80 °C for 1 - 24 h;

[0072] In one embodiment of the present invention, the purification steps are as follows: after treatment with amylase, LR-CDs are prepared by rotary evaporation, ethanol precipitation, centrifugation, dialysis, and drying; ethanol is used for ethanol precipitation, and the addition ratio of the reaction solution to ethanol is 1:1 - 1:10. The cut-off molecular weight of the dialysis bag is 500 - 3000 Da. The drying methods include freeze drying, atmospheric drying, spray drying, drum drying or microwave drying.

[0073] In one embodiment of the present invention, the starch required for the preparation of LR-CDs includes, but is not limited to: potato starch, corn starch, cassava starch, pea starch, wheat starch, rice starch.

[0074] The present invention provides a cassava starch gel with increased gel strength prepared by the above method.

[0075] The present invention also provides a method for regulating the gel strength of starch. The method is as follows: Starch is dispersed in water to form a starch suspension. After adding LR-CDs with a DP greater than 8 to the starch suspension for gelatinization, different addition amounts are used to obtain starch gels with enhanced or weakened gel strength.

[0076] The mass fraction of starch in the starch suspension is 5-50%.

[0077] In one embodiment of the present invention, the addition amount of the LR-CDs, based on the addition amount of starch, is 1%-10% of the dry weight of the starch.

[0078] In one embodiment of the present invention, the conditions for gelatinization are: 90-100°C, 30-60 minutes.

[0079] In one embodiment of the present invention, the starch includes but is not limited to potato starch, corn starch, cassava starch, wheat starch, and pea starch.

[0080] In one embodiment of the present invention, 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), 29%-30% (w / w), 30%-31% (w / w), 31%-32% (w / w), 32%-33% (w / w), 33%-34% (w / w), 34%-35% (w / w), 35%-36% (w / w), 36%-37% (w / w), 37%-38% (w / w), 38%-39% (w / w), 39%-40% (w / w), 40%-41% (w / w), 41%-42% (w / w), 42%-43% (w / w), 43%-44% (w / w), 44%-45% (w / w), 45%-46% (w / w), 46%-47% (w / w), 47%-48% (w / w), 48%-49% (w / w), or 49%-50% (w / w).

[0081] In one embodiment of the present invention, the addition amount of the macrocyclic dextrin (LR-CDs), based on the addition amount of starch, 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) or 9% - 10% (w / w) of the dry weight of starch.

[0082] The present invention provides a starch gel obtained by the above application.

[0083] The present invention also provides the above application, or the above preparation method, or the application of the above cassava starch gel or the above starch gel in the food field.

[0084] In one embodiment of the present invention, the food is canned food. The application of cassava starch gel or the above starch gel in canned food is mainly reflected in its characteristics as a thickener, stabilizer and binder; cassava starch gel or the above starch gel plays an important role in canned food, enhancing the quality and taste of canned food by providing stability and improving the texture of the food.

[0085] In one embodiment of the present invention, the food is frozen food. The high freeze-thaw stability of cassava starch gel or the above starch gel makes it particularly suitable for frozen food because it can maintain the stability of the structure during the freeze-thaw cycle, prevent water loss, and maintain the quality and taste of the food.

[0086] In one embodiment of the present invention, the food is condiments and soup bases.

[0087] In one embodiment of the present invention, the food is sausage, meat and fish products. The application of cassava starch gel or the above starch gel as condiments in meat products such as sausage, meat and fish products is mainly as a thickener, stabilizer and binder.

[0088] In one embodiment of the present invention, the food is dairy products. Cassava starch gel or the above starch gel can be used as a stabilizer to help maintain the product structure and extend the shelf life, such as in the production of yogurt, cheese, etc.

[0089] In one embodiment of the present invention, the food is pearl milk tea. The application of cassava starch in pearl milk tea is mainly used to make the "pearls" in the milk tea, also known as boba or tapioca balls. The pearls are small balls made of cassava starch, which become chewy and Q after being cooked in the milk tea and are an essential ingredient in pearl milk tea. In pearl milk tea, the use of cassava starch adds rich taste layers to the milk tea.

[0090] In one embodiment of the present invention, the food is a beverage or a candy; tapioca starch and its derivatives can improve the processing process of beverages and candies, providing better stability and sensory quality.

[0091] The present invention also provides the above application, or the above preparation method, or the application of the above tapioca starch gel or the above starch gel in the field of chemical products.

[0092] In one embodiment of the present invention, the chemical products include, but are not limited to, adhesives, glues, paper products, and textiles.

[0093] The present invention also provides the above application, or the above preparation method, or the application of the above tapioca starch gel or the above starch gel in biological products or pharmaceuticals.

[0094] In one embodiment of the present invention, the biological products include, but are not limited to, biodegradable plastics.

[0095] Beneficial effects

[0096] (1) The present invention provides a method for preparing LR-CDs using 4-α-glycosyltransferase and its application in increasing the gel strength of tapioca starch. After adding 5% (percentage based on dry starch) of LR-CDs, the gel formed after the gelatinization of tapioca starch has an increased gel hardness, and the effect is significant. This method belongs to adding homologous substances, and the operation is convenient and simple. It neither makes the food have a too sweet or too greasy taste or causes an undesired change in the texture of the food like adding exogenous substances such as salts, sugars, oils, fats, and colloids, nor brings safety hazards like chemical modification. The gel of native tapioca starch is soft and has poor stability. Therefore, this greatly improves the application characteristics of tapioca starch gel and is of great significance for its application in food processing.

[0097] (2) The present invention provides a method for preparing LR-CDs using 4-α-glycosyltransferase and its application in regulating the gel strength of starch. After adding 1% - 10% (percentage based on dry starch) of LR-CDs, the gel formed after the gelatinization of starch can enhance or weaken the hardness of different types of starch gels to varying degrees. This method belongs to adding homologous substances, and the operation is convenient and simple. It neither makes the food have a too sweet or too greasy taste or causes an undesired change in the texture of the food like adding exogenous substances such as salts, sugars, oils, fats, and colloids, nor brings safety hazards like chemical modification. Precise regulation of gel strength can balance the structural strength and functional properties of materials, which is the key to realizing the multi-scenario adaptable application of starch-based materials and is of great significance for its application in food processing. Brief description of the drawings

[0098] Figure 1 : LC-MS identification diagram of the prepared LR-CDs (DP10 - 28).

[0099] Figure 2 : Images of cassava starch gels before and after adding 5% LR-CDs.

[0100] Figure 3 : Effect of adding macrocyclic cyclodextrin on the hardness of cassava starch gels.

[0101] Figure 4 : Effect of adding macrocyclic cyclodextrin on the hardness of potato starch gels.

[0102] Figure 5 : Effect of adding macrocyclic cyclodextrin on the hardness of corn starch gels.

[0103] Figure 6 : Effect of adding macrocyclic cyclodextrin on the hardness of wheat starch gels.

[0104] Figure 7 : Effect of adding macrocyclic cyclodextrin on the hardness of pea starch gels. Detailed implementation manners

[0105] The following are descriptions of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0106] Technical terms:

[0107] Gel strength:

[0108] Gel strength refers to the mechanical properties of gel materials, which is an important indicator for measuring the quality of gel materials. Gel strength can be improved by adjusting the composition and preparation process of gel materials. For example, the strength of the gel can be adjusted by changing the crosslinking degree, structural density, and molecular weight of polymers. In addition, texture parameters such as the hardness and viscosity of the gel are also important components of gel strength.

[0109] Macrocyclic cyclodextrin:

[0110] Macrocyclic cyclodextrins (LR-CDs) are cyclic α-1,4-glucans composed of 9 to hundreds of pyranose glucose units. LR-CDs are mainly obtained by the intramolecular transglycosylation of amylose through the cyclization of cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) or 4-α-glucanotransferase (4αGT, EC 2.4.1.25). Compared with common α, β, γ-cyclodextrins, LR-CD has a larger cavity size and exhibits unique structural and physical-chemical properties, which enable LR-CD to show great application potential in the fields of food, medicine, biology, etc.

[0111] In the present invention, LR-CDs are prepared and added to cassava starch to participate in the gelatinization process together, and the gel strength of the starch gel formed after gelatinization and cooling is enhanced.

[0112] In the present invention, LR-CDs are prepared and added to starch to participate in the gelatinization process together. Different addition amounts of LR-CDs enhance or weaken the gel strength of the starch gel formed after gelatinization and cooling, realizing the regulation of the gel strength of the starch gel.

[0113] It should be noted that any method can be used to prepare the target product LR-CDs in the present invention; the preparation method of the LR-CDs can be any of the following methods:

[0114] a. After gelatinizing the starch, add 4-α-glycosyltransferase for liquefaction reaction. After the reaction ends, simultaneously add debranching enzyme and cyclizing enzyme for synchronous action. Add amylase to the obtained reaction product for purification reaction, and obtain LR-CDs after the purification step.

[0115] b. After gelatinizing the starch, add 4-α-glycosyltransferase for liquefaction reaction. After the reaction ends, first add debranching enzyme to the obtained starch for debranching, then add glycosyltransferase for reaction. Add amylase to the obtained reaction product for purification reaction, and obtain LR-CDs after the purification step.

[0116] c. Using starch as the raw material, after debranching treatment with debranching enzyme, add an appropriate amount of glycosyltransferase, select appropriate addition amount and action time to produce LR-CDs. After inactivating the enzyme and centrifuging, then treat with amylase for a period of time, inactivate the enzyme, centrifuge, rotary evaporate, alcohol precipitate and dialyze, and finally dry to obtain the target product LR-CDs.

[0117] Using any method to prepare the target product LR-CDs, as long as DP is greater than 8, the technical solution of the present invention can be realized.

[0118] In one aspect, the DP range of LR-CDs is 9 to 50.

[0119] In one aspect, the DP of 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.

[0120] In one aspect, the DP of the LR-CDs can 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 to 23, 23 to 24, 24 to 25, 25 to 26, 26 to 27, 27 to 28, 28 to 29, 29 to 30, 30 to 31, 31 to 32, 32 to 33, 33 to 34, 34 to 35, 35 to 36, 36 to 37, 37 to 38, 38 to 39, 39 to 40, 40 to 41, 41 to 42, 42 to 43, 43 to 44, 44 to 45, 45 to 46, 46 to 47, 47 to 48, 48 to 49 or 49 to 50.

[0121] Only the preparation methods of two kinds of LR-CDs are taken as examples below:

[0122] The first preparation method of LR-CDs:

[0123] The method for preparing LR-CDs in the present invention is as follows: using starch as a raw material, after gelatinization, first add debranching enzyme for debranching reaction, then perform secondary gelatinization, after secondary gelatinization, add an appropriate amount of glycosyltransferase for cyclization reaction, select appropriate addition amounts and reaction times to produce LR-CDs, after inactivating the enzyme and centrifuging, then treat with amylase for a period of time, inactivate the enzyme, centrifuge, rotary evaporate, alcohol precipitate and dialyze, and finally dry to obtain the target product LR-CDs.

[0124] In one aspect, when preparing large-ring cyclodextrin (LR-CDs), all starches as substrates are applicable to the present invention. The starch can be derived from any natural source, and "natural" starch is the unmodified form found in nature. Starch is a carbohydrate widely present in plants, mainly synthesized by amyloplasts from glucose molecules and stored in the roots, stems, leaves and seeds of plants. According to different plant sources, the types and properties of starch are also different. The natural source can be cereal starch, legume starch, tuber starch, etc., such as corn, peas, potatoes, sweet potatoes, barley, wheat, rice, cassava, sorghum, lotus root, yam.

[0125] In one aspect, as used herein, the term "pea starch" refers to starch prepared from the seeds of the pea (Pisum Sativum). In one aspect, the pea starch is prepared from the garden pea (Pisum Sativum ssp. Hortense). In another aspect, the pea starch is prepared from the red pea (Pisum Sativum ssp. arvense). In one aspect, the pea starch is prepared from fresh peas. In another aspect, the pea starch is prepared from frozen peas. In one aspect, the pea starch does not include a significant amount of protein. In one aspect, the pea starch includes no more than 5% (w / w) protein. In one aspect, the pea starch does not include a significant amount of fiber. In one aspect, the pea starch includes a amylose content greater than 25% (w / w). In another aspect, the pea starch includes an amylose content greater than 30% (w / w). In a further aspect, the pea starch includes an amylose content greater than 35% (w / w).

[0126] In one aspect, the gelatinization refers to the process in which starch granules change from a solid state to a gel-like or paste-like 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 property of starch in food processing and industrial applications; in an embodiment of the present invention, the gelatinization is carried out by reacting at a certain temperature after adding water; in the present invention, the starch suspension can be gelatinized, and the temperature can be directly raised for gelatinization; different types of starch have different gelatinization temperatures. For example, the gelatinization temperature of corn starch is about between 60°C and 80°C; the gelatinization temperature of potato starch is about between 60°C and 80°C; the gelatinization temperature of wheat starch is about between 60°C and 70°C; the gelatinization temperature of rice starch is about between 70°C and 85°C; the gelatinization temperature of lotus seed starch is about between 70°C and 80°C.

[0127] In a laboratory environment, a water bath is often used. However, in actual industrial production and food processing, as long as the starch is added with water and then the temperature is raised for the gelatinization reaction, the technical solution of the present invention can be achieved.

[0128] In one aspect, the debranching reaction of the starch of the present invention:

[0129] The debranching reaction of starch refers to the process of removing the branch points in amylopectin during the biosynthesis of starch to obtain amylose. This process is of great significance in starch metabolism and industrial applications.

[0130] Factors affecting debranching:

[0131] Structure of Branch Points: Starch is a polysaccharide composed of glucose units linked by α-1,4- and α-1,6-glycosidic bonds. Amylopectin, like glycogen and some types of starch, contains α-1,6-glycosidic bonds as branch points. Role of Debranching Enzymes: Debranching reactions are usually catalyzed by specific enzymes called debranching enzymes (such as pullulanase, EC 3.2.1.41). These enzymes recognize α-1,6-glycosidic bonds and cleave them. Biological Significance of Debranching Reactions: In plants, debranching reactions contribute to the regulation of starch degradation, affecting the final structure and properties of starch. In animals, debranching enzymes assist in the breakdown of 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 has wide applications in industries such as food, textiles, and papermaking. 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: By means of genetic engineering, the activity of starch synthase can be altered, thereby producing starches with different structures in plants, including starches with fewer branches. Regulation of Debranching Reactions: The efficiency and specificity of debranching reactions can be controlled by adjusting conditions such as enzyme concentration, temperature, and pH value.

[0132] In one aspect, the debranching enzyme can be pullulanase, which is a type of starch debranching enzyme named for its ability to specifically hydrolyze pullulan (a polymer of maltotriose linked by α-1,6 glycosidic bonds), belonging to the amylase class, and capable of specifically cleaving the α-1,6 glycosidic bond in the branch point of amylopectin, cutting off the entire branch structure to form amylose; the pullulanase can be commercially available pullulanase, or pullulanase prepared by microbial fermentation, or chemically synthesized pullulanase, and the method used can be any method known in the art.

[0133] Determination of the Enzyme Activity of Pullulanase

[0134] The activity of pullulanase is determined by the 3,5-dinitrosalicylic acid method (3,5-Dinitrosalicylic acid, DNS). Using 10 mg / ml glucose as the standard, a standard curve is made under different dilution conditions.

[0135] Dissolve 1 g of pullulan in 100 mL of phosphate buffer (20 mM, pH 6.0) to prepare a 1% (w / v) substrate. Take 0.9 mL of the substrate and preheat it at 40 °C for 10 min, add 0.1 mL of enzyme solution (0.1 mg / mL), and after reacting for 10 min, add 1.0 mL of DNS to terminate the reaction. After the sample is developed color by boiling water bath for 5 min, immediately cool it in ice water. Measure the absorbance at 540 nm. The amount of enzyme required to catalyze the production of a reducing power equivalent to 1 μmol of glucose per minute is defined as one enzyme activity unit (U).

[0136] In one aspect, the debranching enzyme can be isoamylase, which only hydrolyzes the α-1,6 glycosidic bonds at the branch points of glycogen or amylopectin, cuts off the entire side branches, and forms amylose with different lengths. The isoamylase can be a commercially available isoamylase, or an isoamylase prepared by microbial fermentation, or a chemically synthesized isoamylase, and the method used can be any method well known in the art.

[0137] In one aspect, in the debranching reaction, the addition amount of the debranching enzyme, calculated based on the substrate, is at least 10 U / g of starch.

[0138] In one aspect, in the debranching reaction, the addition amount of the debranching enzyme can be from 600 U / g of starch to 610 U / g of starch, can be from 610 U / g of starch to 620 U / g of starch, can be from 620 U / g of starch to 630 U / g of starch, can be from 630 U / g of starch to 640 U / g of starch, can be from 640 U / g of starch to 650 U / g of starch, can be from 650 U / g of starch to 660 U / g of starch, can be from 660 U / g of starch to 670 U / g of starch, can be from 670 U / g of starch to 680 U / g of starch, can be from 680 U / g of starch to 690 U / g of starch, can be from 690 U / g of starch to 700 U / g of starch, can be from 700 U / g of starch to 710 U / g of starch, can be from 710 U / g of starch to 720 U / g of starch, can be from 720 U / g of starch to 730 U / g of starch, can be from 730 U / g of starch to 740 U / g of starch, can be from 740 U / g of starch to 750 U / g of starch, can be from 750 U / g of starch to 760 U / g of starch, can be from 760 U / g of starch to 770 U / g of starch, can be from 770 U / g of starch to 780 U / g of starch, can be from 780 U / g of starch to 790 U / g of starch, can be from 790 U / g of starch to 800 U / g of starch, and can be from 800 U / g of starch to 1000 U / g of starch.

[0139] In one aspect, the temperature for carrying out the debranching reaction can be from 40 °C to 80 °C.

[0140] In one aspect, the temperature for the debranching 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.

[0141] In one aspect, the time for the debranching reaction is: 1 - 24 h.

[0142] In one aspect, the time for the debranching reaction is: 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h.

[0143] The cyclization reaction is as follows:

[0144] 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 the cyclization reaction involves several key steps: The E region of CGTase has multiple maltose binding sites (MBS), which can bind to the substrate and guide the substrate into the active center of the enzyme.

[0145] In one aspect, 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. The factors affecting the cyclization reaction of cyclodextrin include the type and source of the enzyme, reaction time, temperature, pH value, enzyme dosage, additives, type and concentration of the substrate, degree of pretreatment of the raw material, etc. These factors not only affect the yield of cyclodextrin but also affect the specificity of the product.

[0146] In one aspect, the cyclizing enzyme can be 4-α-glycosyltransferase, cyclodextrin glucosyltransferase.

[0147] In one aspect, 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.

[0148] In one aspect, the cyclodextrin glucosyltransferase can be a commercially available cyclodextrin glucosyltransferase, or a cyclodextrin glucosyltransferase prepared by microbial fermentation, or a chemically synthesized cyclodextrin glucosyltransferase, and any method known in the art can be used.

[0149] In one aspect, in the cyclization reaction, the addition amount of the cyclizing enzyme, calculated based on the substrate, can be: 1 U / g starch, 2 U / g starch, 5 U / g starch, 10 U / g starch, 20 U / g starch, 30 U / g starch, 40 U / g starch, 50 U / g starch, 60 U / g starch, 70 U / g starch, 80 U / g starch, 90 U / g starch, 100 U / g starch.

[0150] In one aspect, in the cyclization reaction, the addition amount of the cyclizing enzyme 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.

[0151] In one aspect, in the cyclization reaction, the addition amount of the cyclase can be 1 U / g starch to 2 U / g starch, 2 U / g starch to 3 U / g starch, 3 U / g starch to 4 U / g starch, 4 U / g starch to 5 U / g starch, 5 U / g starch to 6 U / g starch, 6 U / g starch to 7 U / g starch, 7 U / g starch to 8 U / g starch, 8 U / g starch to 9 U / g starch, 9 U / g starch to 10 U / g starch, 10 U / g starch to 11 U / g starch, 11 U / g starch to 12 U / g starch, 12 U / g starch to 13 U / g starch, 13 U / g starch to 14 U / g starch, 14 U / g starch to 15 U / g starch, 15 U / g starch to 16 U / g starch, 16 U / g starch to 17 U / g starch, 17 U / g starch to 18 U / g starch, 18 U / g starch to 19 U / g starch or 19 U / g starch to 20 U / g starch.

[0152] In one aspect, the temperature for carrying out the cyclization reaction can be 40 °C to 80 °C.

[0153] In one aspect, the temperature for carrying out 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.

[0154] In one aspect, the time for carrying out the cyclization reaction is 1 to 12 h.

[0155] In one aspect, the time for carrying out the cyclization reaction is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h.

[0156] After carrying out the debranching reaction and the cyclization reaction in the present invention, an amylase is added for the purification reaction.

[0157] In one aspect, the amylase can be β-amylase, glucoamylase;

[0158] In one aspect, the β-amylase, also known as starch β-1,4-maltosidase, is one of the amylase classes and is widely present in higher plants such as barley, wheat, sweet potato, soybean, etc. and microorganisms such as the genus Bacillus. The β-amylase can be a commercially available β-amylase, or a β-amylase prepared by microbial fermentation, or a chemically synthesized β-amylase, and the method adopted can be any method well known in the art.

[0159] Method for determining β-amylase activity

[0160] The β-amylase activity level was determined by the 3,5-dinitrosalicylic acid method (DNS method): Take 0.94 mL of 1% soluble starch solution prepared with Na2HPO4-NaH2PO4 buffer (pH 6.0), preheat it at 40 °C for 5 min, add 0.06 mL of β-amylase enzyme solution, mix well, incubate at 40 °C for 15 min, take 0.4 mL of the reaction solution, add 1.2 mL of DNS reagent, heat it in a boiling water bath for 10 min, after cooling to room temperature, take 0.2 mL and add 0.8 mL of deionized water, mix well and measure the OD value at a wavelength of 540 nm. The standard curve was plotted with different concentrations of the 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 an equivalent of 1 mg of maltose per hour under the conditions of pH 6.0 and 40 °C.

[0161] In one aspect, the glucoamylase is a customary name, and its scientific name is α-1,4-glucan glucohydrolace. The glucoamylase can be a commercially available glucoamylase, or a glucoamylase prepared by microbial fermentation, or a chemically synthesized glucoamylase, and any method known in the art can be used.

[0162] In one aspect, the purification steps can be: alcohol precipitation treatment, dialysis, and drying.

[0163] In one aspect, the alcohol precipitation is carried out with absolute ethanol.

[0164] In one aspect, the dialysis is carried out using a dialysis bag with a molecular weight cut-off of 1000 Da to remove small molecular sugars.

[0165] In one aspect, the drying methods include, but are 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.

[0166] In one aspect, any LR-CDs with suitable properties applicable herein are purified by any method known in the prior art before or after any modification or transformation to remove impurities natural to starch or generated during processing.

[0167] In one aspect, the products of the enzymatic preparation of LR-CDs from starch contain not only LR-CDs, but also common cyclodextrins, linear dextrins, and maltooligosaccharides. Therefore, the first step in separating LR-CDs is to remove the non-cyclic components. Since LR-CDs do not have a reducing end and cannot be degraded by amylases that recognize the ends, such as β-amylase, isoamylase, and glucoamylase, using these enzymes to catalyze the LR-CDs product can degrade the linear molecules, thereby preliminarily purifying the cyclic components.

[0168] In one aspect, the purification and separation method is thin-layer chromatography. Thin-layer chromatography is a commonly used separation method, which is simple and easy to perform. However, in the separation and analysis of sugars, the conventional silica gel plates used in TLC are only used for the separation of monosaccharides and oligosaccharides.

[0169] In one aspect, the purification and separation method is high-performance liquid chromatography (HPLC). The key factor in separating LR-CDs by HPLC lies in the selection of the chromatographic column and the mobile phase. The mixture of LR-CDs and oligosaccharides produced after the catalysis of starch needs to be separated using a gel column, and the LR-CDs fraction is collected and then separated using a reverse-phase ODS column.

[0170] In one aspect, the purification of LR-CD is carried out by separation using chromatographic analysis techniques. 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 were separated using a reverse chromatographic column and an amino column respectively; or according to the method of Koizumi et al. (Zheng M Y, 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 chromatographic column, and the methanol eluent with different volume fractions was adjusted to finally obtain LR-CDs products in the range of DP10-11, DP12-20 and DP21-31.

[0171] In one aspect, the addition amount of the amylase used for purification can be 1 U / g starch to 100 U / g, and can be 1 U / g starch, 2 U / g starch, 3 U / g starch, 4 U / g starch, 5 U / g starch, 6 U / g starch, 7 U / g starch, 8 U / g starch, 9 U / g starch, 10 U / g starch, 11 U / g starch, 12 U / g starch, 13 U / g starch, 14 U / g starch, 15 U / g starch, 16 U / g starch, 17 U / g starch, 18 U / g starch, 19 U / g starch, 20 U / g starch, 21 U / g starch, 22 U / g starch, 23 U / g starch, 24 U / g starch, 25 U / g starch, 26 U / g starch, 27 U / g starch, 28 U / g starch, 29 U / g starch or 30 U / g starch.

[0172] In one aspect, in the purification reaction, the addition amount of amylase can be 1 U / g starch to 2 U / g starch, 2 U / g starch to 3 U / g starch, 3 U / g starch to 4 U / g starch, 4 U / g starch to 5 U / g starch, 5 U / g starch to 6 U / g starch, 6 U / g starch to 7 U / g starch, 7 U / g starch to 8 U / g starch, 8 U / g starch to 9 U / g starch, 9 U / g starch to 10 U / g starch, 10 U / g starch to 11 U / g starch, 11 U / g starch to 12 U / g starch, 12 U / g starch to 13 U / g starch, 13 U / g starch to 14 U / g starch, 14 U / g starch to 15 U / g starch, 15 U / g starch to 16 U / g starch, 16 U / g starch to 17 U / g starch, 17 U / g starch to 18 U / g starch, 18 U / g starch to 19 U / g starch, 19 U / g starch to 20 U / g starch, 21 U / g starch to 22 U / g starch, 22 U / g starch to 23 U / g starch, 23 U / g starch to 24 U / g starch, 24 U / g starch to 25 U / g starch, 25 U / g starch to 26 U / g starch, 26 U / g starch to 27 U / g starch, 27 U / g starch to 28 U / g starch, 28 U / g starch to 29 U / g starch or 29 U / g starch to 30 U / g starch.

[0173] In one aspect, the reaction temperature of the amylase for purification can be 40 °C to 80 °C.

[0174] 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.

[0175] In one aspect, the time of the purification reaction is 1 to 24 h.

[0176] In one aspect, the time of the purification reaction is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h.

[0177] The second preparation method of LR-CDs:

[0178] The present invention also provides a method for preparing LR-CDs. In this method, after gelatinizing high-substrate-concentration starch as the substrate, 4-α-glycosyltransferase is added for liquefaction reaction. After the reaction ends, debranching enzyme and cyclase are added simultaneously for synchronous action. After adding amylase to the obtained reaction product for purification reaction and through the purification steps, LR-CDs are obtained.

[0179] Or after gelatinizing the starch, 4-α-glycosyltransferase is added for liquefaction reaction. After the reaction ends, debranching enzyme is first added to the obtained starch for debranching, then glycosyltransferase is added for reaction. After adding amylase to the obtained reaction product for purification reaction and through the purification steps, LR-CDs are obtained.

[0180] In one aspect, the starch can be reacted in the form of a starch suspension, and the mass fraction of starch in the starch suspension is greater than 5%; preferably greater than 10%.

[0181] In one aspect, the starch can be reacted in the form of a starch suspension, and the mass fraction of starch in the starch suspension can be 5% - 6%, 6% - 7%, 7% - 8%, 8% - 9%, 9% - 10%, 10% - 11%, 11% - 12%, 12% - 13%, 13% - 14%, 14% - 15%, 15% - 16%, 16% - 17%, 17% - 18%, 18% - 19%, 19% - 20%, 21% - 22%, 22% - 23%, 23% - 24%, 24% - 25%, 25% - 26%, 26% - 27%, 27% - 28%, 28% - 29% or 29% - 30%.

[0182] In one aspect, the present invention first performs a liquefaction reaction on the gelatinized starch;

[0183] In one aspect, in the liquefaction reaction, the addition amount of 4-α-glycosyltransferase can be 1 U / g starch - 2 U / g starch, 2 U / g starch - 3 U / g starch, 3 U / g starch - 4 U / g starch, 4 U / g starch - 5 U / g starch, 5 U / g starch - 6 U / g starch, 6 U / g starch - 7 U / g starch, 7 U / g starch - 8 U / g starch, 8 U / g starch - 9 U / g starch, 9 U / g starch - 10 U / g starch, 10 U / g starch - 11 U / g starch, 11 U / g starch - 12 U / g starch, 12 U / g starch - 13 U / g starch, 13 U / g starch - 14 U / g starch, 14 U / g starch - 15 U / g starch, 15 U / g starch - 16 U / g starch, 16 U / g starch - 17 U / g starch, 17 U / g starch - 18 U / g starch, 18 U / g starch - 19 U / g starch, 19 U / g starch - 20 U / g starch.

[0184] In one aspect, in step (1), the simultaneous liquefaction reaction during the gelatinization process is as follows: after adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, the temperature is raised to 80-100°C and maintained for 20-60 min.

[0185] In one aspect, after the simultaneous liquefaction reaction during the gelatinization process, the DE value of the starch is controlled to be: 2-8.

[0186] In one aspect, when the liquefying enzyme is α-amylase, the addition amount is: calculated based on the substrate, 1 U / g starch - 20 U / g starch;

[0187] In one aspect, when the liquefying enzyme is cyclodextrin glucosyltransferase, the addition amount is: calculated based on the substrate, 1 U / g starch - 20 U / g starch.

[0188] In one aspect, after the liquefaction reaction of the starch suspension in the present invention, the debranching reaction and the cyclization reaction are started.

[0189] The enzymes added in the debranching reaction and the cyclization reaction and the addition amounts are the same as those in the first preparation method. The difference is that in the second method, the debranching and cyclization are synchronous reactions.

[0190] In the present invention, the reaction conditions for achieving synchronous debranching and cyclization can be: reacting at 50°C for 12 h; can be: reacting at 50°C for 24 h; can be: reacting at 60°C for 12 h; can be: reacting at 60°C for 24 h, etc.

[0191] The conditions for synchronous debranching and cyclization are not limited to the above conditions.

[0192] In one aspect, the temperature for synchronous debranching and cyclization can be 50°C - 80°C.

[0193] In one aspect, the temperature for synchronous 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.

[0194] In one aspect, the time for synchronous debranching and cyclization is: 1 - 48 h.

[0195] In one aspect, the time for simultaneous debranching and cyclization is: 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h.

[0196] In one aspect, after the simultaneous debranching and cyclization of the present invention, amylase is added for a purification reaction; the method of the purification reaction is the same as that of the first preparation method.

[0197] The present invention provides a method for modifying cassava starch: the method is to add large ring dextrins (LR-CDs) with a degree of polymerization (DP) greater than 8 to cassava starch for gelatinization modification.

[0198] In one aspect, the DP range of LR-CDs is 9 to 50.

[0199] In one aspect, the DP of 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.

[0200] In one aspect, the DP of LR-CDs can 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 to 23, 23 to 24, 24 to 25, 25 to 26, 26 to 27, 27 to 28, 28 to 29, 29 to 30, 30 to 31, 31 to 32, 32 to 33, 33 to 34, 34 to 35, 35 to 36, 36 to 37, 37 to 38, 38 to 39, 39 to 40, 40 to 41, 41 to 42, 42 to 43, 43 to 44, 44 to 45, 45 to 46, 46 to 47, 47 to 48, 48 to 49 or 49 to 50.

[0201] In one aspect, the cassava starch can be reacted in the form of a starch suspension, and the mass fraction of starch in the starch suspension is 1 to 30% (w / w).

[0202] In one aspect, the cassava starch can be reacted in the form of a starch suspension, and the mass fraction of the starch in the cassava 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), 23% (w / w), 24% (w / w), 25% (w / w), 26% (w / w), 27% (w / w), 28% (w / w), 29% (w / w) or 30% (w / w).

[0203] In one aspect, the cassava starch can be reacted in the form of a starch suspension, and the mass fraction of the starch in the cassava starch suspension can be 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), 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).

[0204] In one aspect, gelatinization refers to the process in which starch granules change from a solid state to a gel-like or pasty state under the action of heat and water. This process causes significant changes in the physical and chemical properties of starch and is an important characteristic of starch in food processing and industrial applications. In the embodiments of the present invention, the gelatinization is carried out by reacting at a certain temperature after adding water. In the present invention, the starch suspension can be gelatinized by directly raising the temperature. Different types of starch have different gelatinization temperatures. For example, the gelatinization temperature of corn starch is approximately between 60°C and 80°C; the gelatinization temperature of potato starch is approximately between 60°C and 80°C; the gelatinization temperature of wheat starch is approximately between 60°C and 70°C; the gelatinization temperature of rice starch is approximately between 70°C and 85°C; the gelatinization temperature of lotus seed starch is approximately between 70°C and 80°C.

[0205] In a laboratory environment, a water bath is often used. However, in actual industrial production and food processing, as long as water is added to the starch and the temperature is raised for the gelatinization reaction, the technical solution of the present invention can be achieved.

[0206] In one aspect, the addition amount of macrocyclic dextrin (LR-CDs), based on the addition amount of starch, is 1% - 10% (w / w) of the dry basis weight of starch.

[0207] In one aspect, the addition amount of macrocyclic dextrin (LR-CDs), based on the addition amount of starch, 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) of the dry basis weight of starch.

[0208] In one aspect, the addition amount of macrocyclic dextrin (LR-CDs), based on the addition amount of starch, 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) or 9% - 10% (w / w) of the dry basis weight of starch.

[0209] In one aspect, the gelatinization reaction is as follows: after adding macrocyclic dextrin (LR-CDs) to the starch suspension, the temperature is raised to 80 - 100°C and maintained for 20 - 60 min.

[0210] The present invention provides a method for modifying starch: the method is to add macrocyclic dextrin (LR-CDs) with a degree of polymerization (DP) greater than 8 to potato, cassava, corn, wheat, and pea starches for modification.

[0211] In one aspect, the DP range of LR-CDs is 9 - 50.

[0212] In one aspect, 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.

[0213] 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, 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.

[0214] In one aspect, the starch can be gelatinized in the form of a starch suspension, and the mass fraction of the starch in the starch suspension is 5-50% (w / w).

[0215] In one aspect, the starch can be gelatinized in the form of a starch suspension, and the mass fraction of the starch in the starch suspension can be 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 11% (w / w), 12% (w / w), 13% (w / w), 14% (w / w), 15% (w / w), 16% (w / w), 17% (w / w), 18% (w / w), 19% (w / w), 20% (w / w), 21% (w / w), 22% (w / w), 23% (w / w), 24% (w / w), 25% (w / w), 26% (w / w), 27% (w / w), 28% (w / w), 29% (w / w), 30% (w / w), 31% (w / w), 32% (w / w), 33% (w / w), 34% (w / w), 35% (w / w), 36% (w / w), 37% (w / w), 38% (w / w), 39% (w / w), 40% (w / w), 41% (w / w), 42% (w / w), 43% (w / w), 44% (w / w), 45% (w / w), 46% (w / w), 47% (w / w), 48% (w / w), 49% (w / w) or 50% (w / w).

[0216] In one aspect, the starch can be gelatinized in the form of a starch suspension, and the mass fraction of the starch in the starch suspension can be 5-50% (w / w); preferably, the mass fraction of the 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), 20%-21% (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), 29%-30% (w / w), 30%-31% (w / w), 31%-32% (w / w), 32%-33% (w / w), 33%-34% (w / w), 34%-35% (w / w), 35%-36% (w / w), 36%-37% (w / w), 37%-38% (w / w), 38%-39% (w / w), 39%-40% (w / w), 40%-41% (w / w), 41%-42% (w / w), 42%-43% (w / w), 43%-44% (w / w), 44%-45% (w / w), 45%-46% (w / w), 46%-47% (w / w), 47%-48% (w / w), 48%-49% (w / w) or 49%-50% (w / w).

[0217] In one aspect, the addition amount of macrocyclic dextrin (LR-CDs), based on the addition amount of starch, is 1%-10% (w / w) of the dry basis weight of the starch.

[0218] In one aspect, the addition amount of macrocyclic dextrin (LR-CDs), based on the addition amount of starch, 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) of the dry basis weight of the starch.

[0219] In one aspect, the addition amount of macrocyclic dextrin (LR-CDs), based on the addition amount of starch, 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) or 9% - 10% (w / w) of the dry weight of starch.

[0220] In one aspect, the gelatinization reaction is as follows: after adding macrocyclic dextrin (LR-CDs) to the starch suspension, the temperature is raised to 80 - 100 °C and maintained for 20 - 60 min;

[0221] It will be appreciated from the foregoing that the present disclosure can be embodied in various ways, including but not limited to the following:

[0222] Example 1: One solution: A method for enhancing the gel strength of cassava starch. The method is to disperse cassava starch in water to form a cassava starch suspension, add LR-CDs with a DP greater than 8 to the cassava starch suspension for gelatinization, and obtain a cassava starch gel with enhanced gel strength.

[0223] Another solution: A method for regulating the gel strength of starch. The method is to disperse starch in water to form a starch suspension, add LR-CDs with a DP greater than 8 to the starch suspension for gelatinization, and obtain a starch gel with enhanced or weakened gel strength.

[0224] Example 2: According to the method described in Example 1, the preparation method of the LR-CDs can be any of the following methods:

[0225] a. After gelatinizing the starch, add 4-α-glycosyltransferase for liquefaction reaction. After the reaction ends, simultaneously add debranching enzyme and cyclase for synchronous action. Add amylase to the obtained reaction product for purification reaction, and after purification steps, LR-CDs are obtained.

[0226] b. After gelatinizing the starch, add 4-α-glycosyltransferase for liquefaction reaction. After the reaction ends, first add debranching enzyme to the obtained starch for debranching, then add glycosyltransferase for reaction. Add amylase to the obtained reaction product for purification reaction, and after purification steps, LR-CDs are obtained.

[0227] c. Using starch as the raw material, first perform debranching treatment with debranching enzyme, then add an appropriate amount of glycosyltransferase, select appropriate addition amount and action time to produce LR-CDs. After inactivating the enzyme and centrifuging, then treat with amylase for a period of time, inactivate the enzyme, centrifuge, rotary evaporate, precipitate with alcohol and dialyze, and finally dry to obtain the target product LR-CDs.

[0228] Example 3: According to the method described in Example 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.

[0229] Example 4: According to the method described in Example 2 or 3, in method a or b, the mass fraction of starch in the starch suspension is greater than 5%; preferably greater than 10%; the mass fraction can be 5% to 30%; in method c, the mass fraction of starch in the starch suspension is 1% to 10%.

[0230] Example 5: According to any one of the methods described in Examples 2 to 4, the debranching enzyme can be isoamylase, and the isoamylase only hydrolyzes the α-1,6 glycosidic bonds at the branch points of glycogen or amylopectin, cutting off the entire side branches to form amylose with different lengths. The isoamylase can be a commercially available isoamylase, or an isoamylase prepared by microbial fermentation, or a chemically synthesized isoamylase, and the method used can be any method well known in the art; the addition amount of the debranching enzyme, calculated based on the substrate, is at least 100 U / g of starch.

[0231] The cyclase can be 4-α-glycosyltransferase, 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.

[0232] The cyclodextrin glucosyltransferase can be a commercially available cyclodextrin glucosyltransferase, or a cyclodextrin glucosyltransferase prepared by microbial fermentation, or a chemically synthesized cyclodextrin glucosyltransferase, and the method used can be any method well-known in the art;

[0233] In the cyclization reaction, the addition amount of the cyclizing enzyme, calculated based on the substrate, can be: 1 U / g starch to 20 U / g starch.

[0234] Example 6: According to the method described in any one of Examples 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 h; the temperature for the cyclization reaction can be 40°C to 80°C, and the time can be: 1 to 12 h;

[0235] In method c, the simultaneous liquefaction reaction during the gelatinization process means that after adding α-amylase or cyclodextrin glucosyltransferase to the starch suspension, the temperature is raised to 80 - 100°C and maintained for 20 - 60 min; the temperature for the simultaneous debranching and cyclization can be 50°C to 80°C, and the time for the simultaneous debranching and cyclization is: 1 to 48 h.

[0236] Example 7: According to the method described in any one of Claims 1 to 6, add amylase for the purification reaction; the amylase can be β-amylase or glucoamylase.

[0237] Example 8: According to the method described in Example 7, the β-amylase, also known as starch β-1,4-maltosidase, is one of the amylase enzymes and is widely present in higher plants such as barley, wheat, sweet potato, and soybean, as well as microorganisms such as the genus Bacillus. The β-amylase can be a commercially available β-amylase, or a β-amylase prepared by microbial fermentation, or a chemically synthesized β-amylase, and the method used can be any method well-known in the art.

[0238] Example 9: According to the method described in Example 7, the glucoamylase is a customary name, and its scientific name is α-1,4-glucan glucohydrolace. The glucoamylase can be a commercially available glucoamylase, or a glucoamylase prepared by microbial fermentation, or a chemically synthesized glucoamylase, and the method used can be any method well-known in the art.

[0239] Example 10: According to the method described in any one of Examples 1 to 9, the addition amount of the amylase for purification can be 1 U / g starch to 100 U / g, and can be 1 U / g starch, 2 U / g starch, 3 U / g starch, 4 U / g starch, 5 U / g starch, 6 U / g starch, 7 U / g starch, 8 U / g starch, 9 U / g starch, 10 U / g starch, 11 U / g starch, 12 U / g starch, 13 U / g starch, 14 U / g starch, 15 U / g starch, 16 U / g starch, 17 U / g starch, 18 U / g starch, 19 U / g starch, 20 U / g starch, 21 U / g starch, 22 U / g starch, 23 U / g starch, 24 U / g starch, 25 U / g starch, 26 U / g starch, 27 U / g starch, 28 U / g starch, 29 U / g starch or 30 U / g starch.

[0240] Example 11: According to the method described in any one of Examples 1 to 10, in the purification reaction, the addition amount of the amylase can be 1 U / g starch to 2 U / g starch, 2 U / g starch to 3 U / g starch, 3 U / g starch to 4 U / g starch, 4 U / g starch to 5 U / g starch, 5 U / g starch to 6 U / g starch, 6 U / g starch to 7 U / g starch, 7 U / g starch to 8 U / g starch, 8 U / g starch to 9 U / g starch, 9 U / g starch to 10 U / g starch, 10 U / g starch to 11 U / g starch, 11 U / g starch to 12 U / g starch, 12 U / g starch to 13 U / g starch, 13 U / g starch to 14 U / g starch, 14 U / g starch to 15 U / g starch, 15 U / g starch to 16 U / g starch, 16 U / g starch to 17 U / g starch, 17 U / g starch to 18 U / g starch, 18 U / g starch to 19 U / g starch, 19 U / g starch to 20 U / g starch, 21 U / g starch to 22 U / g starch, 22 U / g starch to 23 U / g starch, 23 U / g starch to 24 U / g starch, 24 U / g starch to 25 U / g starch, 25 U / g starch to 26 U / g starch, 26 U / g starch to 27 U / g starch, 27 U / g starch to 28 U / g starch, 28 U / g starch to 29 U / g starch or 29 U / g starch to 30 U / g starch.

[0241] Example 12: According to the method described in any one of Examples 1 to 11, the reaction temperature of the amylase for purification can be 40°C to 80°C.

[0242] Example 13: According to the method described in any one of 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.

[0243] Example 14: According to the method described in any one of Examples 1 to 13, the time of the purification reaction is: 1 to 24 h.

[0244] Example 15: According to the method described in any one of Examples 1 to 14, the time of the purification reaction is: the time of the purification reaction is: 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h.

[0245] Example 16: According to the method described in any one of Examples 1 to 15, the purification steps can be: alcohol precipitation treatment, dialysis, drying; the alcohol precipitation is: alcohol precipitation is carried out with absolute ethanol; the dialysis is carried out with a dialysis bag with a molecular weight cut-off of 1000 Da to remove small molecular sugars; the drying methods include but are not limited to natural drying, hot air drying, vacuum drying, freeze drying, spray drying, drum drying, fluidized bed drying, infrared drying, microwave drying, ultrasonic drying; any LR-CDs with suitable properties applicable to this article are purified by any method known in the prior art before or after any modification or transformation to remove impurities natural to starch or generated during processing.

[0246] Example 17: According to the method described in any one of Examples 1 to 16, the product of enzymatic catalysis of starch to prepare LR-CDs contains not only LR-CDs, but also common cyclodextrins, linear dextrins, and maltooligosaccharides, etc. Therefore, the first step in separating LR-CDs is to remove non-cyclic components. Since LR-CDs do not have a reducing end and cannot be degraded by amylases that recognize the end, such as β-amylase, isoamylase, and glucoamylase, these enzymes can be used to catalyze the LR-CDs product, so that the linear molecules can be degraded, and thus the cyclic components can be preliminarily purified.

[0247] 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 perform. However, in the separation and analysis of sugars, the conventional silica gel plates used in TLC are only used for the separation of monosaccharides and oligosaccharides.

[0248] Example 19: According to the method described in any one of Examples 1 to 18, the purification and separation method is high-performance liquid chromatography (HPLC). The key factors for separating LR-CDs by HPLC lie in the selection of the chromatographic column and the mobile phase. The mixture of LR-CDs and oligosaccharides produced after the catalysis of starch needs to be separated using a gel column, and the LR-CDs fraction is collected, and then separated using a reversed-phase ODS column.

[0249] Example 20: According to the method described in any one of Examples 1 to 20, the purification of LR-CD is to separate by using chromatographic analysis techniques. 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 were separated using a reversed-phase chromatographic column and an amino column respectively; it can also be carried out according to the method of Koizumi et al. (Zheng M Y, 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 chromatographic column, and the methanol eluent with different volume fractions was adjusted to finally obtain LR-CD products in the range of DP10-11, DP12-20 and DP21-31.

[0250] The detection methods involved in the following examples are as follows:

[0251] Identification method of LR-CDs

[0252] The degree of polymerization distribution of LR-CDs was determined by WATERS MALDI SYNAPT Q-TOF MS. The chromatographic column was BEHC18 2.1×150 mmol / L 1.7 μm, and the detection wavelength was 200 - 400 nm. The sample was prepared with ultrapure water at a concentration of 1 mg / mL. The mobile phase was 0.1% formic acid (A) and acetonitrile (B), and the elution program was as follows: 0 - 40 min, 100% A; 40 - 45 min, 70% A and 30% B; 45 - 50 min, 20% A and 80% B; 50 - 55 min, 100% B. The column temperature was 45 °C, the injection volume was 5 μL, and the flow rate was 0.3 mL / min. The ionization mode was electrospray ionization - negative ion mode, and the ion spray voltage was 4 kV. The nebulizing and drying gas was nitrogen, and the collision gas was helium.

[0253] Example 1: Preparation and identification of LR-CDs

[0254] The specific steps are as follows:

[0255] 1. Preparation of LR-CDs

[0256] (1) Gelatinization of starch:

[0257] Weigh a certain amount of potato starch and disperse it in NaAc-HAc buffer solution (20 mM, pH 4.6) to prepare a 5% (w / w) potato starch solution. Stir and heat it in a boiling water bath for gelatinization for 30 min. After the solution is evenly dispersed, cool the starch solution to 60 °C;

[0258] (2) Debranching reaction

[0259] According to the addition amount of adding 700 U (excess) of pullulanase (Bacillus acidopullulyticus, EC 3.2.1.41, CAS: 9075-68-7, purchased from Novozymes, Denmark) per gram of dry starch (calculated based on the potato starch weighed in step (1)), add pullulanase to the gelatinized starch solution obtained in step (1), and enzymatically hydrolyze it in a water bath at 60 °C for 24 h. After the reaction is completed, boil the obtained reaction solution in a boiling water bath for 30 min, centrifuge it at 7000 g for 20 min, then take the supernatant, dry it, grind it, and sieve it to obtain debranched potato starch.

[0260] (3) Secondary gelatinization

[0261] Weigh a certain amount of the debranched potato starch obtained in step (2) and dissolve it in sodium citrate buffer solution (20 mmol / L, pH 6.0) to prepare a 2% (w / w) potato starch solution. Stir and preheat it in a boiling water bath for 30 min, then put it into an autoclave, and keep it at 121 °C for 20 min to make it completely gelatinized. After complete gelatinization, take it out and cool it down to the reaction temperature of 75 °C;

[0262] (4) Cyclization reaction

[0263] Add 4-α-glucosyltransferase (derived from Thermoproteus uzoniensis, NCBI accession number: WP_013679179.1) at 10 U / g starch (based on the potato starch weighed in step (1)) to the reaction system after secondary gelatinization obtained in step (3). After reacting at 75 °C for 10 h, terminate the reaction by boiling water bath for 30 min to obtain a reaction solution;

[0264] (5) Purification of LR-CDs

[0265] Add β-amylase (derived from sweet potato, EC 3.2.1.2, 974 U / mg, CAS: 9000-91-3, purchased from Sigma-Aldrich, USA) at 30 U / g starch (based on the potato starch weighed in step (1)) to the reaction solution to remove the unreacted amylose. After reacting at 50 °C for 12 h, terminate the reaction by boiling water bath heating for 30 min, and centrifuge at a high speed of 8000 g for 30 min to remove the precipitated denatured enzyme protein. Collect the supernatant. After the supernatant is concentrated by rotary evaporation, add 4 volumes of absolute ethanol to the concentrated solution and place it at 4 °C for 12 h. After alcohol precipitation, centrifuge at 8000 g for 10 min to collect the precipitate, dissolve it by soaking with deionized water, dialyze with deionized water for 48 h (cut-off molecular weight 1000 Da), and after freeze-drying, obtain the LR-CD sample.

[0266] 2. Identification method of LR-CDs

[0267] The degree of polymerization distribution of LR-CDs was determined by WATERS MALDI SYNAPT Q-TOF MS, and the results are as Figure 1 shown, and LR-CDs with DP 10-28 were prepared.

[0268] Example 2: Effect of LR-CDs on the texture of cassava starch gel

[0269] Disperse cassava starch (CAS) in water to prepare a starch suspension with a mass fraction of 7% (w / w), and add LR-CD (DP 10-28) samples with different mass fractions (1%, 3%, 5%, 7%, 10% w / w based on the dry weight of starch) prepared in Example 1 to it. Stir magnetically for 2 min to make the starch granules disperse evenly. Put it into a water bath with magnetic force, stir magnetically at 95 °C for 30 min to completely gelatinize it, and then quickly pour it into a disposable plastic petri dish with a diameter of 35 mm and a height of 10 mm, and refrigerate at 4 °C for 24 h to form a starch gel.

[0270] The texture properties of starch gels were measured using a TA-XT plus texture analyzer at room temperature (25 °C). 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; deformation: 50%; interval between two presses: 5 s; trigger force: 5.0 g. The test for each sample was repeated 3 times.

[0271] Using 7% cassava starch (weighing 2 g of cassava starch and adding water to prepare a starch suspension with a mass fraction of cassava starch of approximately 7%) as a control, the same treatment was carried out according to the above steps, with the difference that LR-CDs were not added and the rest of the operations remained unchanged.

[0272] The results are shown in Table 1.

[0273] Table 1 Texture parameters of cassava starch gels

[0274]

[0275] Note: The data in the table are expressed as mean ± standard deviation, and different lowercase letters in the same column indicate significant differences (P < 0.05).

[0276] According to the data in Table 1 and Figure 3 it can be seen that for the gels formed by adding LR-CDs (1% - 10%) to cassava starch, with the increase in the addition amount of LR-CDs, the hardness of the cassava starch gels shows a trend of first increasing and then decreasing. The addition of 1%, 3%, and 5% of LR-CDs increased the hardness of the cassava starch gels from 137.01 g to 249.28 g, 319.50 g, and 402.23 g respectively, which were increased by 1.82 times, 2.33 times, and 2.94 times respectively, and the gel strength was enhanced significantly. It can also be clearly observed from Figure 2 that the gel formed by refrigerating native cassava starch at 4 °C for 24 h showed a collapsed state and could not form a fixed shape, while the cassava starch gel added with LR-CDs formed a solid cylindrical gel with a stable shape. However, when the addition amount exceeded 5%, the hardness decreased and the gel strength showed a downward trend. Therefore, different addition amounts of LR-CDs can regulate the strength of cassava starch gels to different degrees.

[0277] Example 3: Effect of LR-CDs on the texture of potato starch gels

[0278] Disperse potato starch (PS) in water to prepare a starch suspension with a concentration of 7% (w / w), and add samples of LR-CDs (DP10 - 28) with different mass fractions (1%, 3%, 5%, 7%, 10% w / w based on the dry weight of starch) prepared in Example 1. Stir magnetically for 2 min to evenly disperse the starch granules. Place it in a water bath with a magnetic stirrer, stir magnetically at 95 °C for 30 min to completely gelatinize it, and then quickly pour it into a disposable plastic Petri dish with a diameter of 35 mm and a height of 10 mm, and refrigerate at 4 °C for 24 h to form a starch gel.

[0279] Use a TA-XT plus texture analyzer to measure the texture properties of the starch gel at room temperature of 25 °C. 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; Deformation amount 50%, interval between two presses 5 s, trigger force 5.0 g. Each sample is tested 3 times.

[0280] Take 7% potato starch (weigh 2 g of potato starch and add water to prepare a starch suspension with a potato starch mass fraction of about 7%) as a control, and perform the same treatment according to the above steps, except that LR-CDs are not added and the rest of the operations remain unchanged.

[0281] The results are shown in Table 2.

[0282] Table 2 Texture parameters of potato starch gel

[0283]

[0284] Note: The data in the table are expressed as mean ± standard deviation, and different lowercase letters in the same column indicate significant differences (P < 0.05).

[0285] According to Table 2 and Figure 4 the data in it, it can be seen that for the gel formed after adding LR-CDs to potato starch, the hardness of the gel will also increase. The addition of 1%, 3%, 5%, and 7% of LR-CDs increases the hardness of the potato starch gel from 308.32 g to 334.43 g, 352.03 g, 376.68 g, and 373.64 g respectively, and the hardness increases by 8.47%, 14.18%, 22.17%, and 21.18% respectively. The improvement effect is not as significant as that of cassava starch gel; when the addition amount of LR-CDs reaches 10%, the hardness of the gel will decrease to 315.37 g. Therefore, different addition amounts of LR-CDs can regulate the strength of potato starch gel to different degrees.

[0286] Example 4: Effect of LR-CDs on the texture of corn starch gel

[0287] Corn starch (CS) was dispersed in water to prepare a starch suspension with a concentration of 7% (w / w), and LR-CDs (DP10 - 28) samples with different mass fractions (1%, 3%, 5%, 7%, 10% w / w based on the dry weight of starch) prepared in Example 1 were added thereto. Magnetic stirring was carried out for 2 min to uniformly disperse the starch granules. It was placed in a water bath with magnetic force, and magnetic stirring was carried out at 95 °C for 30 min to completely gelatinize it, and then it was quickly poured into a disposable plastic petri dish with a diameter of 35 mm and a height of 10 mm, and refrigerated at 4 °C for 24 h to form a starch gel.

[0288] The texture properties of the starch gel were measured using a TA-XT plus texture analyzer at room temperature of 25 °C. Test conditions: probe model: P36R; measurement height: 15 mm; speed before measurement: 0.5 mm / s; test speed: 0.5 mm / s; speed after measurement: 0.5 mm / s; deformation amount 50%, interval between two depressions 5 s, trigger force 5.0 g. The test for each sample was repeated 3 times.

[0289] Taking 7% corn starch (weighing 2 g of corn starch and adding water to prepare a starch suspension with a corn starch mass fraction of about 7%) as a control, it was treated in the same manner as the above steps, with the difference that LR-CDs were not added and the rest of the operations remained unchanged.

[0290] The results are shown in Table 3.

[0291] Table 3 Texture parameters of corn starch gel

[0292]

[0293] Note: The data in the table are expressed as mean ± standard deviation, and different lowercase letters in the same column indicate significant differences (P < 0.05).

[0294] According to Table 3 and Figure 5 the data in it, when LR-CDs were added to corn starch, when the addition amount was low, the hardness of the corn starch gel did not show a significant change; when the addition amount reached 5%, the hardness of the gel increased from 542.53 g to 670.49 g, with a hardness increase of 23.59%; but when the addition amount of LR-CDs continued to increase, the hardness would decrease. With the addition of 7% and 10% LR-CDs, the hardness of the corn starch gel decreased to 426.52 g and 379.73 g. Therefore, LR-CDs with different addition amounts can enhance or weaken the strength of corn starch gel to different degrees.

[0295] Example 5: Effect of LR-CDs on the texture of wheat starch gel

[0296] Disperse wheat starch (WS) in water to prepare a starch suspension with a concentration of 7% (w / w), and add samples of LR-CDs (DP10 - 28) with different mass fractions (1%, 3%, 5%, 7%, 10% w / w based on the dry weight of starch) prepared in Example 1. Stir magnetically for 2 min to evenly disperse the starch granules. Place it in a water bath with a magnetic stirrer, stir magnetically at 95 °C for 30 min to completely gelatinize it, and then quickly pour it into a disposable plastic petri dish with a diameter of 35 mm and a height of 10 mm, and refrigerate at 4 °C for 24 h to form a starch gel.

[0297] Use a TA-XT plus texture analyzer to measure the texture properties of the starch gel at room temperature of 25 °C. 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; deformation amount 50%, interval between two presses 5 s, trigger force 5.0 g. Each sample is tested 3 times.

[0298] Use 7% wheat starch (weigh 2 g of wheat starch and add water to prepare a starch suspension with a wheat starch mass fraction of approximately 7%) as a control, and perform the same treatment according to the above steps, with the difference that LR-CDs are not added and the rest of the operations remain unchanged.

[0299] The results are shown in Table 4.

[0300] Table 4 Texture parameters of wheat starch gel

[0301]

[0302] Note: The data in the table are expressed as mean ± standard deviation, and different lowercase letters in the same column indicate significant differences (P < 0.05).

[0303] According to Table 4 and Figure 6 the data in it, it can be seen that as the addition amount of LR-CDs increases, the hardness of the wheat starch gel shows a trend of first increasing and then decreasing. The addition of 1% LR-CDs increases the hardness of the wheat starch gel from 696.41 g to 878.87 g, with a hardness increase of 26.20%, and the chewiness increases from 485.01 g to 622.88 g; while the addition of 3% and 5% LR-CDs reduces the hardness of the wheat starch gel from 696.41 g to 644.76 g and 373.44 g. Therefore, the addition of 1% LR-CDs can improve the gel strength of wheat starch, but when the addition amount exceeds 1%, continuing to add LR-CDs will cause the gel strength to decrease.

[0304] Example 6: Effect of LR-CDs on the texture of pea starch gel

[0305] Disperse pea starch (PEA) in water to prepare a starch suspension with a concentration of 7% (w / w), and add samples of LR-CDs (DP10 - 28) with different mass fractions (1%, 3%, 5%, 7%, 10% w / w based on the dry weight of starch) prepared in Example 1. Stir magnetically for 2 min to evenly disperse the starch granules. Place it in a water bath with magnetic stirring, stir magnetically at 95 °C for 30 min to completely gelatinize it, and then quickly pour it into a disposable plastic petri dish with a diameter of 35 mm and a height of 10 mm, and refrigerate at 4 °C for 24 h to form a starch gel.

[0306] Use a TA-XT plus texture analyzer to measure the texture properties of the starch gel at room temperature of 25 °C. 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; deformation amount 50%, interval between two depressions 5 s, trigger force 5.0 g. Each sample is tested 3 times.

[0307] Take 7% pea starch (weigh 2 g of pea starch and add water to prepare a starch suspension with a pea starch mass fraction of about 7%) as a control, and perform the same treatment according to the above steps, with the difference that LR-CDs are not added and the rest of the operations remain unchanged.

[0308] The results are shown in Table 5.

[0309] Table 5 Texture parameters of pea starch gel

[0310]

[0311] Note: The data in the table are expressed as mean ± standard deviation, and different lowercase letters in the same column indicate significant differences (P < 0.05).

[0312] According to Table 5 and Figure 7 the data, it can be seen that the hardness of the pea starch gel is significantly higher than that of the other four starches. This is because the amylose content in pea starch is relatively high, and amylose forms a rigid three-dimensional network through double helix winding after gelatinization, providing high-strength mechanical support. With the increase in the addition amount of LR-CDs, the hardness of the pea starch gel shows a trend of first increasing and then decreasing. When the addition amount is 1%, the strength of the pea starch gel is the largest, and if the addition continues, the gel strength will decrease. Therefore, LR-CDs with different addition amounts can regulate the strength of pea starch gel to different degrees.

[0313] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Application of macrocyclic dextrins (LR-CDs) with a degree of polymerization (DP) greater than 8 in increasing the strength of starch gels.

2. The application according to claim 1, wherein The application is as follows: Starch is dispersed in water to form a starch suspension, and the LR-CDs are added to the starch suspension for gelatinization to obtain a starch gel with enhanced gel strength; preferably, 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, 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; Preferably, 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 addition amount of the LR-CDs, based on the addition amount of starch, is 1% - 10% (w / w) of the dry basis weight of starch; Preferably, the addition amount of the macrocyclic dextrins (LR-CDs), based on the addition amount of starch, 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) of the dry basis weight of starch; Preferably, the conditions for gelatinization are: 90 - 100 °C, heating for 30 - 60 min to completely gelatinize the starch.

3. Application of macrocyclic dextrins (LR-CDs) with a degree of polymerization (DP) greater than 8 in regulating the strength of starch gels.

4. The application according to claim 3, characterized in that, The application is as follows: starch is dispersed in water to prepare a starch suspension, and after adding the LR-CDs to the starch suspension for gelatinization, with different addition amounts, starch gels with enhanced or weakened gel strength are obtained; Preferably, 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, 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; Preferably, the mass fraction of starch in the starch suspension is 5-50% (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), 20%-21% (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), 29%-30% (w / w), 30%-31% (w / w), 31%-32% (w / w), 32%-33% (w / w), 33%-34% (w / w), 34%-35% (w / w), 35%-36% (w / w), 36%-37% (w / w), 37%-38% (w / w), 38%-39% (w / w), 39%-40% (w / w), 40%-41% (w / w), 41%-42% (w / w), 42%-43% (w / w), 43%-44% (w / w), 44%-45% (w / w), 45%-46% (w / w), 46%-47% (w / w), 47%-48% (w / w), 48%-49% (w / w) or 49%-50% (w / w); Preferably, the addition amount of the LR-CDs, based on the addition amount of starch, is 1% to 10% (w / w) of the dry basis weight of starch; Preferably, the addition amount of the macrocyclic dextrin (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) or 9% to 10% (w / w) of the dry basis weight of starch; Preferably, the gelatinization conditions are: 90 to 100 °C, heating for 30 to 60 min to completely gelatinize the starch.

5. A method for preparing a cassava starch gel with enhanced starch gel strength, characterized in that, The method is to disperse tapioca starch in water to form a starch suspension, add LR-CDs with a DP greater than 8 to the starch suspension for gelatinization, and obtain a tapioca starch gel with enhanced gel strength; The mass fraction of tapioca starch in the starch suspension is 5% to 30%; Preferably, the addition amount of the LR-CDs, based on the addition amount of tapioca starch, is 1% to 10% of the dry basis weight of tapioca starch; Preferably, the gelatinization conditions are: 90 to 100 °C, 30 to 60 min; Preferably, the mass fraction of starch in the starch suspension is 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w), 15% to 16% (w / w), 16% to 17% (w / w), 17% to 18% (w / w), 18% to 19% (w / w), 19% to 20% (w / w), 21% to 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); Preferably, the addition amount of the macrocyclic dextrin (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) of the dry basis weight of starch; 6. A method for regulating the strength of starch gel, characterized in that, The method is to disperse starch in water to form a starch suspension, add LR-CDs with a DP greater than 8 to the starch suspension for gelatinization, and different addition amounts are added to obtain a starch gel with enhanced or weakened gel strength; The mass fraction of starch in the starch suspension is 5% to 50%; Preferably, the addition amount of the LR-CDs, based on the addition amount of starch, is 1% to 10% of the dry basis weight of starch; Preferably, the conditions for gelatinization are: 90 - 100 °C, 30 - 60 min; Preferably, the starch includes, but is not limited to, potato starch, corn starch, tapioca starch, wheat starch, and pea starch; 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), 29% - 30% (w / w), 30% - 31% (w / w), 31% - 32% (w / w), 32% - 33% (w / w), 33% - 34% (w / w), 34% - 35% (w / w), 35% - 36% (w / w), 36% - 37% (w / w), 37% - 38% (w / w), 38% - 39% (w / w), 39% - 40% (w / w), 40% - 41% (w / w), 41% - 42% (w / w), 42% - 43% (w / w), 43% - 44% (w / w), 44% - 45% (w / w), 45% - 46% (w / w), 46% - 47% (w / w), 47% - 48% (w / w), 48% - 49% (w / w), or 49% - 50% (w / w); Preferably, the addition amount of the macrocyclic dextrin (LR-CDs), based on the addition amount of starch, 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), or 9% - 10% (w / w) of the dry weight of starch.

7. According to the application described in any one of claims 1 to 4 or the method described in claim 5 or 6, characterized in that, The preparation method of the LR-CDs is as follows: after gelatinizing the starch, pullulanase is added for debranching, after debranching, glycosyltransferase is added for cyclization, and after cyclization, amylase is added for purification to obtain LR-CDs; Preferably, the pullulanase includes, but is not limited to: pullulanase, isoamylase; Preferably, the conditions for debranching are: adding 10 - 1000 U / g of starch pullulanase and enzymatically hydrolyzing at 40 - 80 °C for 1 - 24 h; Preferably, the glycosyltransferase includes, but is not limited to: 4-α-glycosyltransferase, cyclodextrin glucosyltransferase; Preferably, the conditions for cyclization are: adding glycosyltransferase at 1-100 U / g of starch and enzymatically hydrolyzing for 1-12 h at 40-80 °C; Preferably, the amylases include, but are not limited to: β-amylase, glucoamylase; Preferably, the conditions for amylase treatment are: adding amylase at 1-100 U / g of starch and enzymatically hydrolyzing for 1-24 h at 40-80 °C; Preferably, the purification step is to obtain LR-CDs after amylase treatment, followed by centrifugation, rotary evaporation, alcohol precipitation, centrifugation, dialysis and drying; Preferably, the starch includes, but is not limited to: potato starch, corn starch, tapioca starch, wheat starch, pea starch.

8. According to the application according to any one of claims 1 to 4 or the method according to claim 5 or 6, characterized in that, The preparation method of the LR-CDs includes the following steps: (1) Adding liquefying enzyme to the starch suspension and synchronously performing a liquefaction reaction during gelatinization; the mass fraction of starch in the starch suspension is greater than 5%; preferably greater than 10%, more preferably greater than 20%; (2) Adding a debranching enzyme and a cyclizing enzyme to the starch obtained in step (1) simultaneously for reaction to obtain a reaction product; or adding a debranching enzyme to the starch obtained in step (1) first for a debranching reaction, and then adding a cyclizing enzyme for reaction to obtain a reaction product; (3) Adding an amylase to the reaction product obtained in step (2) for reaction to prepare LR-CDs; Preferably, the mass fraction of starch in the starch suspension is 5%-30%; Preferably, in step (1), the liquefying enzyme includes, but is not limited to: α-amylase, 4-α-glycosyltransferase or cyclodextrin glucosyltransferase; when the liquefying enzyme is 4-α-glycosyltransferase, the addition amount is: calculated based on the substrate, 1 U / g of starch - 20 U / g of starch; the addition amount of the liquefying enzyme is: calculated based on the substrate, 1 U / g of starch - 20 U / g of starch; Preferably, in step (1), the simultaneous liquefaction reaction during gelatinization means that after adding α-amylase, 4-α-glycosyltransferase or cyclodextrin glucosyltransferase to the starch suspension, the temperature is raised to 80-100 °C and maintained for 20-60 min; Preferably, after the simultaneous liquefaction reaction during gelatinization, the DE value of the starch is controlled to be: 2-8; Preferably, the addition amount of the liquefying enzyme is: 1 U / g of starch - 20 U / g of starch; Preferably, the liquefying enzyme is 4-α-glycosyltransferase; the debranching enzymes include, but are not limited to: pullulanase, isoamylase, amylopectinase; the cyclizing enzymes include, but are not limited to: 4-α-glycosyltransferase, cyclodextrin glucosyltransferase; the addition amount of the debranching enzyme is, calculated based on the substrate, adding at least 100 U / g of starch of the debranching enzyme; the addition amount of the cyclizing enzyme is: calculated based on the substrate, 1 U / g of starch - 100 U / g of starch; Preferably, in step (2), the reaction conditions are: reacting at 50 °C - 80 °C for: 1-48 h; Preferably, in step (3), the amylase is β-amylase or glucoamylase; the addition amount of the amylase is 1 U / g starch to 100 U / g starch calculated based on the substrate; the reaction temperature after adding the amylase is 30°C to 60°C, and the reaction time is 1 to 48 h.

9. The starch gel obtained by the application according to any one of claims 1 to 4 or the method according to claim 5 or 6.

10. The application according to any one of claims 1 to 4, or the method according to claim 5 or 6, or the application or method according to claim 7 or 8, or the application of the starch gel according to claim 9 in the preparation of food, chemical products, biological products, and pharmaceuticals, characterized in that, The food includes, but is not limited to, frozen food, easy-to-swallow food, seasonings, soups, sausages, dairy products, meat, fish products, pearls in pearl milk tea, beverages, and candies; Preferably, the chemical products include, but are not limited to, adhesives, glues, paper products, and textiles; the biological products include, but are not limited to, biodegradable plastics.

Images