A highly branched cyclodextrin and its preparation process, application and product

Abstract

The present invention provides a novel highly branched cyclodextrin and its preparation process, application and product, belonging to the field of biomedical technology. On the basis of highly branched cyclodextrin, the present invention increases the branch content of highly branched cyclodextrin by more than 10% through the synergistic action of 4,6-α-glycosyltransferase and branching enzyme, shortens the average length of the branch by more than 20%, and does not produce additional small molecular weight sugars at the same time, the product yield is high, and almost no reducing sugar is contained. The novel highly branched cyclodextrin prepared by the present invention has a molecular weight of 50000-80000 Da, an α-1,6-glycosidic bond content of 5.5-6.5%, a reducing sugar content of <1%, and is not prone to Maillard reaction. The novel highly branched cyclodextrin with this structural feature has an anti-fatigue effect in exercise and physical activity, can effectively improve athletic performance, and delay the occurrence of fatigue.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a highly branched cyclodextrin and a preparation process, application and product thereof. Background Art

[0002] Carbohydrates play a very important role in sports and physical activities. Supplementing carbohydrates can effectively improve sports performance and delay the occurrence of fatigue. Depending on the function, the type of carbohydrates suitable for sports supplementation is also different.

[0003] Some carbohydrates are suitable for building up glycogen reserves before exercise to provide energy for exercise. For example, eating foods rich in carbohydrates, including rice and pasta, can also be consumed in the form of beverages, such as the dextrin starch obtained by treating starch with acid at 110-140°C as described in patent CN1142232A. These are all conducive to the synthesis of glycogen. However, this process is relatively slow and is generally taken one or several days before exercise.

[0004] Due to different sports scenarios, it is often necessary to quickly replenish carbohydrates before or during exercise to maintain blood sugar concentration and the carbohydrate oxidation rate required for exercise and delay the occurrence of fatigue. Glucose, fructose, sucrose, maltodextrin and mixtures of these substances have been used as a source of carbohydrates for rapid replenishment during exercise. The monosaccharides and disaccharides are limited in practical applications due to their high sweetness and osmotic pressure. Maltodextrin is a potential source of carbohydrate supplements in sports drinks due to its relatively low sweetness and osmotic pressure. However, maltodextrin degrades quickly in the body, which can easily cause blood sugar fluctuations and cannot meet the needs of long-term continuous energy supply.

[0005] Highly branched cyclodextrin (HBCD) is a new type of maltodextrin (patent EP0710674B1, US9562247B2), which is produced from corn starch through the cyclization reaction of branching enzymes. This process forms a ring structure, and the molecular weight is larger than that of ordinary maltodextrin, the osmotic pressure is lower, and the gastric emptying rate is faster. It can be made into a beverage for carbon water supplementation (patent JP2003169642A). However, the effect of HBCD in exercise is not very obvious. In order to maintain the glucose level in the body for a longer time and meet the energy needs during exercise, the branching ratio of HBCD, that is, the α-1,6 glycosidic bond ratio, can be further increased to reduce its degradation rate in the body. Patent US6861519B2 uses α-amylase, β-amylase, glucoamylase, α-glucosyltransferase, etc. to degrade α-1,4 glycosidic bonds to increase the branching content. However, this method will produce a large amount of small molecule sugars, which need to be further separated and purified, such as the ultrafiltration membrane separation technology used in this patent. This method has low raw material utilization efficiency and product yield, and separation and purification further increases production costs. Summary of the invention

[0006] In view of the above-mentioned deficiencies, the present invention provides a highly branched cyclodextrin and its preparation process, application and product. Based on highly branched cyclodextrin, the present invention increases the branch content of highly branched cyclodextrin by more than 10% and shortens the average length of branches by more than 20% through the synergistic action of 4,6-α-glycosyltransferase and branching enzyme, while not additionally producing small molecular weight sugars, and the product yield is high and contains almost no reducing sugar. The highly branched cyclodextrin prepared by the present invention has a molecular weight of 50,000-80,000 Da, an α-1,6-glycosidic bond content of 5.5-6.5%, and a reducing sugar content of <1%. The highly branched cyclodextrin with this structural feature has an anti-fatigue effect in sports and physical activities, can effectively improve athletic performance, and delay the occurrence of fatigue.

[0007] The technical solution of the present invention includes:

[0008] In one aspect, the present invention provides a highly branched cyclodextrin, wherein the molecular weight of the highly branched cyclodextrin is 50000-80000 Da, the content of α-1,6-glycosidic bonds is 5.5-6.5% of the total amount of α-1,6-glycosidic bonds and α-1,4-glycosidic bonds, and the reducing sugar content is <1%;

[0009] Preferably, the molecular weight of the highly branched cyclodextrin is 65000-75000 Da.

[0010] Preferably, the content of α-1,6-glycosidic bonds in the highly branched cyclodextrin is 5.5-6.0% of the total amount of α-1,6-glycosidic bonds and α-1,4-glycosidic bonds.

[0011] Specifically, the highly branched cyclodextrin is compounded with a substance containing an amino group, and no Maillard reaction occurs during high-temperature sterilization or long-term storage, and no color change occurs.

[0012] Preferably, the amino group-containing substance includes one or more of amino acids, polypeptides, and proteins.

[0013] Specifically, the preparation process of the highly branched cyclodextrin comprises: adding 4,6-α-glycosyltransferase and branching enzyme to carbohydrates to carry out a reaction.

[0014] More specifically, the carbohydrate is starch or dextrin containing branches formed by α-1,6-glycosidic bonds.

[0015] Preferably, the carbohydrate comprises highly branched cyclodextrin and / or waxy corn starch.

[0016] Further preferably, the concentration of the highly branched cyclodextrin and / or waxy corn starch is 5%-30%.

[0017] Still more preferably, the concentration of the highly branched cyclodextrin and / or waxy corn starch is 10%-20%.

[0018] More specifically, the added amount of the 4,6-α-glucose transferase is 0.01%-10% (w / w) of carbohydrates.

[0019] Preferably, the added amount of the 4,6-α-glucose transferase is 1%-2% (w / w) of the carbohydrates.

[0020] More specifically, the added amount of the branching enzyme is 0.01%-5% (w / w) of the carbohydrate.

[0021] Preferably, the branching enzyme is added in an amount of 0.1% (w / w) of the carbohydrate.

[0022] More specifically, the branching enzyme includes glycogen branching enzyme and / or starch branching enzyme.

[0023] Preferably, the branching enzyme is Branchzyme, purchased from Univar Solutions, catalog number 2012342.

[0024] Specifically, the mass ratio of the 4,6-α-glycosyltransferase fermentation broth to the branching enzyme is 0.1-10:1.

[0025] Preferably, the mass ratio of the 4,6-α-glycosyltransferase fermentation broth to the branching enzyme is 10:1.

[0026] Specifically, the reaction temperature is 35-65°C, and the reaction time is 1-24h.

[0027] Preferably, the reaction temperature is 60° C. and the reaction time is 4 h.

[0028] Specifically, the preparation process also includes: enzyme inactivation, sterilization and drying.

[0029] Preferably, the enzyme inactivation comprises inactivation at 90-100° C. for 5-10 min.

[0030] Further preferably, the enzyme inactivation comprises inactivation at 95° C. for 5 min.

[0031] Preferably, the sterilization includes ultra-high temperature instantaneous sterilization.

[0032] Further preferably, the ultra-high temperature instantaneous sterilization process is: 137° C. for 10-15 seconds.

[0033] Preferably, the drying is spray drying.

[0034] On the other hand, the present invention provides a process for preparing the highly branched cyclodextrin.

[0035] In another aspect, the present invention provides the use of the highly branched cyclodextrin in the preparation of food, health care products or medicines.

[0036] Specifically, the food or health product helps to relieve physical fatigue, assists in improving memory or enhances immunity.

[0037] Specifically, the medicine is used to prevent diseases caused by excessive fatigue.

[0038] Preferably, the excessive fatigue includes physical fatigue or psychological fatigue;

[0039] The physical fatigue is manifested as slow movements, insensitivity, decreased coordination, irritability and restlessness, etc.

[0040] The mental fatigue includes inattention, memory impairment, difficulty in understanding and reasoning, and slow and inaccurate mental activities.

[0041] In yet another aspect, the present invention provides a food comprising the highly branched cyclodextrin.

[0042] Specifically, the food includes but is not limited to candy, soy milk, yogurt, milk, juice, canned food, biscuits, chocolate, cakes, cream, cheese, sour cream, milk powder, formula milk powder, ice cream, jam, puree, preserved fruit, bread, egg rolls, protein drinks, solid drinks, lactic acid bacteria drinks, carbonated drinks, coffee, jelly, protein bars, energy bars, energy gel or puffed food.

[0043] Preferably, the food comprises protein beverage, solid beverage, lactic acid bacteria beverage, carbonated beverage, coffee, soy milk, yogurt, milk or juice.

[0044] In another aspect, the present invention provides a health product, comprising the above-mentioned highly branched cyclodextrin.

[0045] Specifically, the health care product also includes conventional supplementary materials for health care foods.

[0046] Preferably, the conventional supplementary materials of health food include but are not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids or nutritional enhancers.

[0047] Specifically, the health products include but are not limited to tea products, granules, medicinal wine, capsules, fruit juice, fruit vinegar, granules, fermented dairy products, fermented cereal products, fermented bean products, powders, honey pastes or meal replacement powders.

[0048] Preferably, the health care products include tea products, granules, medicinal wine, fruit juice, and fruit vinegar.

[0049] In yet another aspect, the present invention provides a medicine comprising the above-mentioned highly branched cyclodextrin.

[0050] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0051] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, adhesives, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0052] Specifically, the dosage form of the drug includes, but is not limited to, a gastrointestinal dosage form and a parenteral dosage form according to the administration method.

[0053] Preferably, the dosage form of the drug is a dosage form for administration through the gastrointestinal tract.

[0054] Further preferably, the dosage form for administration via the gastrointestinal tract includes but is not limited to tablets, powders, granules, solutions, capsules, emulsions, suspensions or oils.

[0055] The technical effects achieved by the present invention are:

[0056] The highly branched cyclodextrin prepared by the preparation process provided by the present invention has an average molecular weight of 50,000-80,000 Da, a branch content of 5.5-8%, extremely low content of small molecular weight sugar and reducing sugar (DE value <1), is not prone to Maillard reaction, and is not prone to color change during high-temperature sterilization or long-term storage of sports drinks. The highly branched cyclodextrin has an anti-fatigue effect in sports and physical activities, can effectively improve sports performance, and delay the occurrence of fatigue. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The average molecular weights of commercially available highly branched cyclodextrin CD1 and highly branched cyclodextrin CD2 were compared for size exclusion chromatography (SEC).

[0058] Figure 2 The abundance ratio of α-1,4 linkage and α-1,6 linkage in the sample was measured for 1H-NMR.

[0059] Figure 3 The color changes of sports drinks containing glucose before (left test tube) and after (right test tube) high temperature sterilization.

[0060] Figure 4 The color changes of sports drinks containing ordinary maltodextrin before (left test tube) and after (right test tube) high-temperature sterilization.

[0061] Figure 5 The color changes of sports drinks containing CD2 before high-temperature sterilization (left test tube) and after high-temperature sterilization (right test tube).

[0062] Figure 6 The weight-bearing swimming time for mice. DETAILED DESCRIPTION

[0063] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments; the commercially available highly branched cyclodextrin used in the present invention is not limited to Cluster Dextrin, 4,6-α-glycosyltransferase is not limited to the source of Lactobacillus reuteri 121 bacteria, nor is it limited to Bacillus subtilis fermentation broth, and branching enzyme is not limited to Branchzyme. The Cluster Dextrin, Bacillus subtilis fermentation broth, and Branchzyme described are for illustration and are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0064] The experimental materials used in the present invention are as follows:

[0065] 1. Cluster Dextrin: purchased from Glico, item number 110713823.

[0066] 2. Waxy corn starch: purchased from Dezhou Dacheng, item number 20231018152.

[0067] 3. Branchzyme: purchased from Univar Solutions, product number 2012342.

[0068] 4. 4,6-α-glycosyltransferase: fermentation broth expressed by Bacillus subtilis. Specific method:

[0069] (1) The gene encoding 4,6-α-glycosyltransferase (BRENDA:EC2.4.1.B34) from Lactobacillus reuteri 121 strain was synthesized (Uniprot # Q5SBM0) and inserted into the PstI / BamHI restriction enzyme site in the expression vector pTA-aeaP (Suzhou Junji Gene Technology Co., Ltd.), and the resulting plasmid was then transferred into Bacillus subtilis ATCC 6051a-164S. For the pTA-aeaP vector, B. subtilis ATCC 6051a-164S and the method of plasmid transfection, refer to Engineering Bacillus subtilis ATCC 6051a for the production of recombinant catalases. Minghua Ji et al. J Ind Microbiol Biotechnol. 2021 Jul 1;48(5-6). Specifically, the obtained plasmid was digested with PciI or BsaI to generate linearized dsDNA and transformed into 164S. Transformants were selected on LB plates containing erythromycin to obtain Bacillus subtilis containing the 4,6-α-glycosyltransferase gene. Sequencing confirmed that the heterologous gene had been correctly inserted.

[0070] (2) A single colony of Bacillus subtilis containing the 4,6-α-glycosyltransferase gene was inoculated into 50 mL of LB medium in a 250 mL flask and cultured at 37°C with gentle shaking at 200 rpm for 48 hours. The fermentation broth was collected and centrifuged at 4,000 × g for 10 minutes at 4°C to collect the supernatant, which contained 4,6-α-glycosyltransferase.

[0071] (3) The supernatant was filtered through a PES material 0.2 μm 90 mm vacuum filter cup (MyBio-Zhuoxuan, catalog number VFPPES222500F), and the filtrate was used for the enzyme catalysis reaction of the present invention.

[0072] Example 1 Preparation process of highly branched cyclodextrin CD2

[0073] 1. Enzyme catalyzed reaction: Commercially available highly branched cyclodextrin (Cluster Dextrin, referred to as CD1) was prepared into a 20% aqueous solution, 1% of the raw material weight of 4,6-α-glycosyltransferase fermentation broth and 0.1% of the raw material weight of branch enzyme (Branchzyme) were added, and the reaction was carried out at 60°C for 4 hours.

[0074] 2. Enzyme inactivation: Heat to 95°C and keep for 5 minutes.

[0075] 3. Ultra-high temperature instantaneous sterilization: 137°C, 10-15 seconds.

[0076] 4. Spray drying.

[0077] Example 2 Determination of average molecular weight

[0078] The average molecular weight of the polymer was determined by high performance liquid chromatography (HPLC) equipped with a differential refractive index detector (RID) and a size exclusion chromatography column (SEC). Chromatographic conditions: TSKgel G4000 PWxl column, column temperature 30°C, mobile phase water, flow rate 1 mL / min. Dextran molecular weight standard materials were produced by the Chinese Academy of Metrology and are numbered GBW(E)050005 (Mw = 12600 Da), GBW(E)050006 (Mw = 63300 Da), and GBW(E)050007 (Mw = 102000 Da).

[0079] Dissolve the samples to be tested (highly branched cyclodextrin CD2, commercially available high-branched cyclodextrin CD1) and dextran molecular weight standard substances in water to prepare 1% solutions, inject them for chromatographic analysis, and record the retention time (Rt) of the main peak. Draw a linear fitting standard curve of the retention time (Rt) and the logarithm of the molecular weight of dextran standards with different molecular weights. The logarithm of the molecular weight of the sample is calculated on the standard curve based on the retention time (Rt) of the sample, and then take the antilogarithm to obtain the average molecular weight of the sample.

[0080] The results of the test are as follows Figure 1 As shown in the figure, the average molecular weight of the commercially available highly branched cyclodextrin CD1 is about 97,000 Da, and the average molecular weight of the highly branched cyclodextrin CD2 prepared by the preparation process provided by the present invention is about 69,000 Da. It should be noted that due to the existence of experimental errors, the molecular weights of the highly branched cyclodextrin CD2 obtained in multiple experiments are distributed between 50,000 and 80,000 Da, mainly distributed between 60,000 and 75,000 Da. The average molecular weight of CD2 is 29% smaller than that of CD1, and no small molecular weight sugar is produced.

[0081] Example 3 Determination of the abundance ratio of different heterogeneous connection types

[0082] Dextrin is a polymer formed by glucose molecules connected by α-1,4 and α-1,6 bonds, of which α-1,6 bonds constitute branches in dextrin. The branch content of dextrin can be obtained by measuring the ratio of α-1,4 bonds to α-1,6 bonds in the molecule.

[0083] Dissolve the previously dried sample in deuterated water (D2O) and sonicate to ensure that the sample solution is evenly mixed. Record the 1H-NMR spectrum of the sample solution using a high-field NMR instrument (e.g., 500 MHz or higher). Identify the peaks corresponding to the α-1,4-linked isomeric protons of the glucose units in the sample molecule (around 5.4–5.6 ppm) and the peaks corresponding to the α-1,6-linked isomeric protons (around 4.9–5.1 ppm). Integrate the peak areas of the α-1,4 and α-1,6 protons, and the relative peak areas represent the abundance of each type of linkage. Calculate the ratio of α-1,4 linkages:α-1,6 linkages. The branching content is the percentage of α-1,6 linkages to the sum of α-1,4 linkages and α-1,6 linkages.

[0084] like Figure 2 As shown in the 1H-NMR spectrum and Table 1, the α-1,4 connection: α-1,6 connection in the highly branched cyclodextrin CD2 is 16.09, and the branch content accounts for 5.9%. The α-1,4 connection: α-1,6 connection in the commercially available highly branched cyclodextrin CD1 is 17.74, and the branch content accounts for 5.3%. The α-1,4 connection: α-1,6 connection in ordinary maltodextrin is 22.72. The branch content (i.e., the abundance of α-1,6 connection) in the highly branched cyclodextrin CD2 is 10% more than that in the commercially available highly branched cyclodextrin CD1, and 40% more than that in ordinary maltodextrin. The average molecular weight of the product obtained by the preparation process provided by the present invention is reduced, and the branch content is increased, which can be inferred that the branch length is shortened.

[0085] Table 1 Abundance ratio and branching content of α-1,4 linkage and α-1,6 linkage

[0086]

[0087] Example 4 Determination of reducing sugar content

[0088] Reducing sugar refers to reducing sugars, including glucose, fructose, galactose, lactose, maltose, maltooligosaccharides, etc. The determination of reducing sugar content in samples refers to 6.3 DE value determination in GB / T 20882.6-2021 "Quality Requirements for Starch Sugars Part 6: Maltodextrin". This method uses the redox reaction of reducing sugars with Fehling's reagent, determines the reaction endpoint with methylene blue indicator, and directly titrates the reducing sugar content. The DE value is the percentage of reducing sugars (measured in glucose) in the dry matter of the sample.

[0089] The measurement results show that the preparation process provided by the present invention does not produce new reducing sugars, and the reducing sugar content of the product (CD2) is <1%.

[0090] Example 5 Maillard reaction detection

[0091] In addition to carbohydrates, sports drinks usually also include branched-chain amino acids or peptides or proteins containing amino acids. Reducing sugars will react with amino acids to produce a brown color, affecting the sensory experience. Maillard reaction detection method: prepare a solution containing 20% ​​carbohydrates (glucose, ordinary maltodextrin, or the product CD2 obtained by the preparation process provided by the present invention), 2% branched-chain amino acids BCAA, and 0.3% sodium chloride, and place it in an autoclave at 120°C for 45 minutes. Observe the color change.

[0092] Figure 3 The color change of sports drinks containing glucose before and after high temperature sterilization. Figure 4 The color change of sports drinks containing ordinary maltodextrin before and after high temperature sterilization. Figure 5 The color changes of the sports drink containing the product CD2 obtained by the preparation process provided by the present invention before and after high temperature sterilization. Only CD2 has no obvious color change. This shows that the product CD2 obtained by the preparation process provided by the present invention is not easy to undergo Maillard reaction because it contains almost no reducing sugar, and is not easy to change color during high temperature sterilization or long-term storage of sports drinks.

[0093] Example 6 Anti-fatigue effect test

[0094] Test method for the anti-fatigue effect of carbohydrates in the weight-bearing swimming experiment of mice: refer to the test method for relieving physical fatigue in the "Health Food Function Test and Evaluation Method (2023 Edition)". In the experiment, the solvent is purified water sterilized by high-pressure steam, and different test products (glucose, ordinary maltodextrin, high-branched cyclic dextrin CD2 obtained by the preparation process of the present invention, and commercially available high-branched cyclic dextrin CD1) are weighed in appropriate amounts and prepared into a 0.1 g / mL dosing solution with the solvent. On the day of the experiment, 6-8 week-old C57BL / 6J mice weighing 18-22g were gavaged with 10 mL / kg body weight of solvent or different test products (dose equivalent to 1g / kg body weight). 30 minutes after gavage, mice with 5% body weight of lead-free tin wire at the base of the tail were placed in a swimming box for swimming. The water depth of the swimming box is not less than 30cm, and the water temperature is 25℃±1.0℃. When the mouse sinks underwater after swimming for a period of time and fails to rise to the surface within 7s, the required time is recorded, which is the weight-bearing swimming time of the mouse.

[0095] like Figure 6As shown, 30 minutes after gavage of each test substance, the weighted swimming time of mice in the G1 solvent group (pure water) and the G2 group (glucose) was similar. At the same test dose, the weighted swimming time of mice in the G3 group (ordinary maltodextrin), G4 group (CD2), and G5 group (CD1) increased to varying degrees, extending by 12.9%, 29.2%, and 15.5% respectively compared with G1. Among them, the weighted swimming time of mice in the G4 group (CD2) was the longest, with significant statistical differences compared with the G1 solvent group and G2 (glucose). Compared with G1, no statistical differences were detected in the remaining test groups except G4 (CD2). It shows that the product (CD2) obtained by the preparation process route provided by the present invention has a significant anti-fatigue effect compared with commercially available high-branched cyclodextrin (CD1), ordinary maltodextrin, glucose and other carbohydrates, and can effectively improve exercise performance and delay the occurrence of fatigue.

[0096] Example 7 Preparation of highly branched cyclodextrin using waxy corn starch as raw material

[0097] The raw materials of this process are not limited to commercially available highly branched cyclodextrins, but waxy corn starch can also be used. The only difference between Example 7 and Example 2 is that the enzyme catalyzed reaction in step 1 is different. The specific process is as follows: Enzyme catalyzed reaction: waxy corn starch is prepared into a 10% suspension, heated to 95°C for gelatinization for 10 minutes, cooled to 60°C, 1% of the raw material weight of 4,6-α-glycosyltransferase fermentation broth and 0.5% of the raw material weight of branch enzyme (Branchzyme) are added, and the reaction is carried out at 60°C for 4 hours.

[0098] Referring to the methods of Example 2 and Example 3, the average molecular weight and the abundance ratio of different isomeric connection types of the prepared highly branched cyclodextrin were measured. The measurement results are shown in Table 2.

[0099] Table 2 Determination of highly branched cyclodextrins prepared from waxy corn starch

[0100]

[0101] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A highly branched cyclodextrin, characterized in that: The molecular weight of the highly branched cyclodextrin is 50000-80000Da, the content of α-1,6-glycosidic bonds is 5.5-6.5% of the total amount of α-1,6-glycosidic bonds and α-1,4-glycosidic bonds, and the reducing sugar content is <1%; The preparation process of the highly branched cyclodextrin comprises: adding 4,6-α-glycosyltransferase and branching enzyme to carbohydrates for reaction; the carbohydrates are starch or dextrin containing branches formed by α-1,6-glycosidic bonds.

2. The highly branched cyclodextrin according to claim 1, characterized in that The molecular weight of the highly branched cyclodextrin is 65000-75000 Da.

3. The highly branched cyclodextrin according to claim 1, characterized in that The content of α-1,6-glycosidic bonds in the highly branched cyclodextrin is 5.5-6.0% of the total amount of α-1,6-glycosidic bonds and α-1,4-glycosidic bonds.

4. The highly branched cyclodextrin according to claim 1, characterized in that The carbohydrates include highly branched cyclodextrin and / or waxy corn starch.

5. The highly branched cyclodextrin according to claim 1, characterized in that The branching enzyme includes glycogen branching enzyme and / or starch branching enzyme.

6. The highly branched cyclodextrin according to claim 1, characterized in that The added amount of the 4,6-α-glycosyltransferase is 0.01%-10% (w / w) of the carbohydrates; the added amount of the branching enzyme is 0.01%-5% (w / w) of the carbohydrates.

7. The highly branched cyclodextrin according to claim 1, characterized in that The reaction temperature is 35-65°C, and the reaction time is 1-24h.

8. The highly branched cyclodextrin according to claim 1, characterized in that The highly branched cyclodextrin is compounded with a substance containing an amino group, and no Maillard reaction occurs during high-temperature sterilization or long-term storage, and no color change occurs.

9. The highly branched cyclodextrin according to claim 8, characterized in that The amino group-containing substance includes one or more of amino acids, polypeptides and proteins.

10. The process for preparing the highly branched cyclodextrin according to any one of claims 1 to 9, characterized in that: The preparation process comprises: adding 4,6-α-glycosyltransferase and branching enzyme to carbohydrates for reaction; the carbohydrates are starch or dextrin containing branches formed by α-1,6-glycosidic bonds.

11. Use of the highly branched cyclodextrin according to any one of claims 1 to 9 in the preparation of foods, health products or medicines.

12. The use according to claim 11, characterized in that: The food or health product is helpful to relieve physical fatigue, assist in improving memory or enhance immunity.

13. A food, characterized in that The food contains the highly branched cyclodextrin according to any one of claims 1 to 9.

14. The food according to claim 13, characterized in that The food includes candy, soy milk, yogurt, milk, juice, canned food, biscuits, chocolate, cakes, cream, cheese, sour cream, milk powder, formula milk powder, ice cream, jam, puree, preserved fruit, bread, egg rolls, protein drinks, solid drinks, lactic acid bacteria drinks, carbonated drinks, coffee, jelly, protein bars, energy bars, energy gel or puffed food.

15. A health product, characterized in that: The health product contains the highly branched cyclodextrin described in any one of claims 1 to 9.

16. The health product according to claim 15, characterized in that: The health care products also include conventional supplementary materials for health care foods.

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