Amylopectin-based cyclic glucan and method for processing the same

Abstract

The present disclosure discloses an amylopectin-based cyclic glucan and the processing method for the same, and belongs to the technical field of food processing. The present disclosure uses a starch as a raw material, prepares an amylopectin-based cyclic glucan through sugar chain degradation grading and glycosidase-catalyzed trans-glycoside technology, and can be used as a steady-state carrier material for food active factors. The method of the disclosure has advantages of green environmental protection, high processing yield and low cost. The prepared product has high branching degree, special large ring structure and good water solubility, and can be used for steady-state delivery and active protection of natural functional substances, involving nutritional food, medicine, daily chemicals and other fields.

Claims

1. A method for processing an amylopectin-based cyclic glucan, comprising: dispersing a defatted starch into a solvent to obtain a 1 g/mL to 5 g/mL starch suspension, wherein the dispersing step comprises: suspending 10 to 25 g of the defatted starch in 6 to 10 mL of absolute ethanol, then adding 10 to 100 mL of an acid catalyst solution comprising an acid catalyst to catalyze degradation of the defatted starch, and reacting at 20 to 60° C. for 30 to 120 minutes, thereby obtaining a starch degradation product; dissolving the starch degradation product into a buffer solution, adding a trans-glycosidase preparation to the starch degradation product, wherein the trans-glycosidase preparation comprises a microorganism-derived trans-glycosidase obtained by fermentation of archaea or bacteria, wherein a ratio of sugar chain branching activity to depolymerization activity of the trans-glycosidase preparation is greater than 30, and wherein the trans-glycosidase preparation is from Calditerricolayamamurae UTM801, CGMCC 6185, Streptococcus thermophiles ATCC 14485, Thermomus thermophiles ATCC 33923, or Aeropyrum pernix K1; heating to inactivate the enzyme in the trans-glycosidase preparation; and isolating the amylopectin-based cyclic glucan, wherein a size of a cyclic structure in the amylopectin-based cyclic glucan is DP 19 to DP 50, wherein a ratio of α-1,6 glycosidic bond in the amylopectin-based cyclic glucan is 5.0% to 7.0%, and wherein a molecular weight of the amylopectin-based cyclic glucan is 3 kDa to 9 kDa.

2. The method according to claim 1, wherein an amount of the trans-glycosidase preparation added in the dissolving step is: 600 to 1000 U of the trans-glycosidase preparation per 10 to 25 g of the defatted starch.

3. The method according to claim 1, wherein in the dispersing step, the acid catalyst is prepared as an aqueous solution with a pH value of 2.5 to 4.0 for catalysis, and a volume ratio of the added amount of the acid catalyst aqueous solution to the defatted starch suspension is 10 to 100:6 to 10.

4. The method according to claim 1, wherein in the dissolving step, the starch degradation product is dissolved in the buffer solution to prepare a solution with a mass concentration of 2% to 30%.

5. The method according to claim 1, wherein in the dissolving step, before adding the trans-glycosidase preparation, the buffer solution in which the starch degradation product is dissolved is heated to 60 to 80° C., and then the trans-glycosidase preparation is added, maintaining the temperature and reacting for 8 to 16 hours after adding the trans-glycosidase preparation.

6. The method according to claim 1, wherein a method for activation culture of archaea or bacteria comprises the following steps: taking a bacterial solution stored in a glycerol tube and inoculating it into a sterilized seed LB medium for culture under sterile conditions; and the fermentation comprises the following steps: inoculating a seed solution after activation culture into a fermentation LB medium, incubating in a constant temperature shaker to a bacterial concentration of OD.sub.600 of 0.6, centrifuging at 10,000 rpm for 15 minutes, discarding the supernatant to collect a bacterial cell, and obtaining the trans-glycosidase preparation by steps of lyophilization and pulverization.

7. The method of claim 1, wherein a molecular weight of the amylopectin-based cyclic glucan is 3 kDa to 7 kDa.

8. The method of claim 1, wherein the acid catalyst is any one or more of phosphoric acid, boric acid, sulfonic, acid, and sulfate.

9. The method of claim 1, wherein the buffer solution is phosphate buffer.

10. The method of claim 1, wherein the cyclic structure is composed of 6.5% α-1,6 glycosidic bonds and 93.5% α-1,4 glycosidic bonds, and the average size is DP21.

11. The method of claim 1, wherein a yield is between 6.0% and 20.0%.

12. The method of claim 1, wherein the acid catalyst is boric acid.

13. The method of claim 1, wherein the acid catalyst is phosphoric acid and the trans-glycosidase preparation is from Thermomus thermophiles.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1. MALDI-TOF-MS mass spectrum of an amylopectin-based cyclic glucan.

(2) FIG. 2. Three-dimensional conformation of an amylopectin-based cyclic glucan.

(3) FIG. 3. Cyclic structure configuration of an amylopectin-based cyclic glucan.

(4) FIG. 4. The particle size distribution of β-carotene-amylopectin-based cyclic glucan in Example 1.

DETAILED DESCRIPTION

(5) The content of the present disclosure will be further clarified below with reference to examples, but the content protected by the present disclosure is not only limited to the following examples.

(6) Particle size measurement: the sample to be measured is prepared into a 0.1% (w/v) solution, and the particle size distribution is measured with a Malvern Nano ZS analyzer at 25° C.

(7) Solubility measurement 20 mg of inclusion is weighed accurately and dissolved in 1 mL of deionized water, equilibrates at room temperature for 12 hours in the dark, and is centrifuged at 4° C. (3000 rpm, 5 minutes) to remove insoluble substances. 0.2 mL of the centrifugal solution is taken and added 4 times the volume of absolute ethanol, vortexed for 15 minutes, and centrifuged (10,000 rpm, 5 minutes) and separated to extract the phytochemical ingredients and starch. Take the supernatant and measure the absorbance with a UV spectrometer, and calculate the solubility by substituting the absorbance into the standard curve equation.

(8) Loading ratio calculation: with reference to the soluble phytochemical content (W) and soluble starch mass (M) obtained by a solubility measurement, and a loading ratio calculation formula is as followed loading ratio (%)=W/M×100.

(9) CaCO.sub.2 cell membrane permeability measurement: cell membrane permeability is a mass percentage of phytochemicals in CO.sub.2 cells and a basal part of a lower layer of a isolation cavity to phytochemicals initially added to a upper layer of cells, and a Millicell-ERS electronic voltmeter is used to measure a transepithelial resistance value inside and outside a cell monolayer culture cavity, so as to monitor the tightness between epithelial cells and determine a integrity of a cell monolayer.

(10) Preparation of Trans-Glycosidase Preparations

(11) A. plant-derived trans-glycosidase preparation: it is obtained by extracting from growing cereal grain endosperms, weighing 100 g of growing cereal grains, adding 300 mL phosphate buffer solution (pH 7.2, 50 mM), and obtaining a crude enzyme solution by homogenizing, filtering and centrifuging, which is then separated and purified by ion exchange column and gel chromatography, and obtaining an enzyme preparation by collecting and lyophilizing the active ingredients. Wherein the endosperm of plant cereal grains comprises: endosperm of rice grains, endosperm of wheat grains, endosperm of corn grains, endosperm of sorghum grains and the like.

(12) B. microorganism-derived trans-glycosidase preparation: using archaea or bacteria screened from nature via steps such as activation culture and fermentation to produce enzymes, wherein strain activation: taking a bacterial solution stored in a glycerol tube and inoculating it into a sterilized 250 mL erlenmeyer flask with 100 mL seed LB medium, incubating at 37° C. for 12 hours. Fermentation culture: inoculating with an inoculation amount of 2% (v/v) into a 250 mL erlenmeyer flask containing 100 mL fermentation LB medium under sterile conditions. After inoculation, incubating on a 37° C. constant temperature shaker until a bacterial concentration of OD600=0.6, and centrifuging at 10,000 rpm for 15 minutes, discarding the supernatant to collect a bacterial cell, and an enzyme preparation is obtained by steps such as lyophilization and pulverization. The microorganism comprises: Bacillus stearothermophilus ATCC 7953, Calditerricolayamamurae UTM801 CGMCC 6185, Streptococcus thermophilus ATCC 14485, Thermomus thermophiles ATCC33923, Aeropyrumpernix K1 (available from the Japan Institute of Industrial Technology).

Example 1

(13) Weighing 25 g of a defatted waxy corn starch and suspending it in 10 mL of absolute ethanol, adding 100 mL of a boric acid solution (pH 2.5) and reacting at 40° C. for 120 minutes, neutralizing a pH value, grading precipitation, washing and drying after the reaction is completed;

(14) Dissolving the obtained starch degradation product in 100 mL of a phosphate buffer solution (pH 7.0) to prepare a solution with a mass concentration of 2%, placing in a 70° C. water bath and heating for 30 minutes, and then adding 600 U of rice-derived trans-glycosidase preparation (sugar chain branching activity/depolymerization activity=52) and maintaining the temperature and reacting for 16 hours, heating and inactivating enzymes, centrifuging, and drying the obtained supernatant under vacuum to obtain the target product of an amylopectin-based cyclic glucan.

(15) The average molecular weight of the obtained amylopectin-based cyclic glucan is 4200 Da, in which a size of the cyclic structure is DP 21, a ratio of α-1,6 glycosidic bonds is 6.0%, and a yield is 12.7%.

Example 2

(16) Weighing 20 g of a defatted waxy rice starch and suspending it in 8 mL of absolute ethanol, adding 80 mL of a hydrochloride solution (pH 4.0) and reacting at 50° C. for 90 minutes, neutralizing a pH value, grading precipitation, washing and drying after the reaction is completed;

(17) Dissolving the obtained starch degradation product in 70 mL of a phosphate buffer solution (pH 7.0) to prepare a solution with a mass concentration of 15%, placing in a 70° C. water bath and heating for 40 minutes, and then adding 800 U of Bacillus stearothermophilus-derived trans-glycosidase preparation (sugar chain branching activity/depolymerization activity=31) and maintaining the temperature and reacting for 12 hours, heating and inactivating enzymes, centrifuging, and drying the obtained supernatant under vacuum to obtain the target product.

(18) The average molecular weight of the obtained amylopectin-based cyclic glucan is 3500 Da, in which a size of the cyclic structure is DP 20, a ratio of α-1,6 glycosidic bonds is 5.2%, and a yield is 9.7%.

Example 3

(19) Weighing 25 g of a defatted waxy potato starch and suspending it in 6 mL of absolute ethanol, adding 60 mL of a phosphoric acid solution (pH 3.0) and reacting at 60° C. for 60 minutes, neutralizing a pH value, grading precipitation, washing and drying after the reaction is completed;

(20) Dissolving the obtained starch degradation product in 50 mL of a phosphate buffer solution (pH 7.0) to prepare a solution with a mass concentration of 10%, placing in a 70° C. water bath and heating for 35 minutes, and then adding 700 U of Thermomus thermophiles-derived trans-glycosidase preparation (sugar chain branching activity/depolymerization activity=75) and maintaining the temperature and reacting for 10 hours, heating and inactivating enzymes, centrifuging, and drying the obtained supernatant under vacuum to obtain the target product. Thermomus thermophiles-derived is Thermus thermophiles CGMCC 6186.

(21) The average molecular weight of the obtained amylopectin-based cyclic glucan is 7850 Da, in which a size of the cyclic structure is DP 35, a ratio of α-1,6 glycosidic bonds is 6.6%, and a yield is 18.1%.

Example 4 Application of the Product Obtained in Examples 1-3 as a Carrier

(22) Dissolving the amylopectin-based cyclic glucan obtained in Examples 1-3 in purified water to prepare a solution with a concentration of 0.5 mg/mL; dissolving β-carotene in absolute ethanol to make a mass concentration of 0.2 mg/mL; adding β-carotene solution to the main amylopectin-based cyclic glucan solution at a ratio of 5:1, placing it in a water bath at 40° C. and stirring for 2 hours at a speed of 4000 rpm, and then homogenizing at 15000 rpm for 1 minutes; placing it in an ultrasonic action device, and a treatment is performed at 0° C. for 12 minutes with a power controlled at 200 W; centrifuging, drying the obtained supernatant under vacuum to obtain the corresponding β-carotene-amylopectin-based cyclic glucan inclusion.

(23) The results of the obtained β-carotene-amylopectin-based cyclic glucan inclusions are shown in Table 1.

(24) TABLE-US-00001 TABLE 1 Performance results of β-carotene-amylopectin-based cyclic glucan inclusions prepared from different amylopectin-based cyclic glucans. CaCO.sub.2 cell membrane Solubility in permeability Average Loading water (increased (increased particle size ratio multiples over multiples over (nm) (%) uninclusion) uninclusion) Example 1 275 1.2 130 4.1 Example 2 268 1.5 150 4.5 Example 3 293 1.1 124 3.9

(25) The uninclusion refers to pure β-carotene.

Comparative Embodiment 1

(26) Referring to Example 1, the trans-glycosidase preparation is replaced with 4-α-glycosyltransferase, and other conditions remained unchanged to prepare a starch product.

(27) Including the Following Process:

(28) Weighing 25 g of a defatted waxy corn starch and suspending it in 10 mL of absolute ethanol, adding 100 mL of a boric acid solution (pH 2.5) and reacting at 40° C. for 120 minutes, neutralizing a pH value, grading precipitation, washing and drying after the reaction is completed;

(29) Dissolving the obtained starch degradation product in 100 mL of a phosphate buffer solution (pH 7.0) to prepare a solution with a mass concentration of 2%, placing in a 70° C. water bath and heating for 30 minutes, and then adding 600 U of 4-α-glycosyltransferase (sugar chain branching activity/depolymerization activity=52) and maintaining the temperature and reacting for 16 hours, heating and inactivating enzymes, centrifuging, and drying the obtained supernatant under vacuum to obtain the target product of an amylopectin-based cyclic glucan.

(30) The average molecular weight of the obtained amylopectin-based cyclic glucan is 37500 Da, in a ratio of α-1,6 glycosidic bonds is 4.2%, without cyclic structure.

Comparative Embodiment 2

(31) Dissolving 20 mg of maltohexaose and 200 mg of glucose-1-1 phosphate in 100 nM citric acid buffer solution (pH7.0) containing 5 mM adenosine phosphate and 20 UD-alcohol, adding 1 mg of acidifying enzyme, and reacting at 30° C. for 2 hours. Centrifuging the reaction solution, treating the supernatant at 100° C. for 5 minutes, and centrifuging to remove a denaturing enzyme protein. Adding 50 U of glucoamylase in the supernatant, and the precipitate contained 30 mg of cyclic glucan only containing α-1,4-glucoside bonds, the obtained cyclic glucan has a cyclic structure free of α-1,6 glucoside bonds.

Example 5

(32) Dispersing a defatted corn starch in a solvent to obtain a 1 g/mL starch suspension, and adding an acid catalyst to perform a starch degradation reaction; after the degradation is completed, dissolving the obtained starch in a buffer, and adding an archaea-derived trans-glycosidase preparation (sugar chain branching activity/depolymerization activity=41) and reacting for 10 hours to obtain an amylopectin-based cyclic glucan with an average molecular weight of 4500 Da, in which the cyclic structure is composed of 6.5% α-1,6 glycosidic bonds and 93.5% α-1,4 glycosidic bonds, and the average size is DP21.

(33) Dissolving the obtained starch samples in water to prepare a solution with a concentration of 0.5 mg/mL; dissolving β-carotene in absolute ethanol to make a solution with a mass concentration of 0.2 mg/mL; adding the β-carotene solution to the main amylopectin-based cyclic glucan solution at a ratio of 5:1, placing it in a water bath at 40° C. and stirring for 2 hours at a speed of 4000 rpm, and then homogenizing at 15000 rpm for 1 minutes; placing it in an ultrasonic action device, and a treatment is performed at 0° C. for 12 minutes with a power controlled at 200 W; centrifuging, drying the obtained supernatant under vacuum to obtain a high nutritional quality of β-carotene-amylopectin-based cyclic glucan inclusion.

(34) An average particle size of the obtained inclusion is 280 nm, and a loading rate of the β-carotene is 1.4%; a solubility in water is 120 times higher than that of pure β-carotene, and a permeability of CaCO.sub.2 cell membrane is 4.2 times higher than that of pure β-carotene.

Example 6

(35) Dispersing a defatted rich starch in a solvent to obtain a 5 g/mL starch suspension, and adding an acid catalyst to perform a starch degradation reaction; after the degradation is completed, dissolving the obtained starch in a buffer, and adding a plant-derived trans-glycosidase preparation (sugar chain branching activity/depolymerization activity=32.1) and reacting for 8 hours to obtain an amylopectin-based cyclic glucan with an average molecular weight of 3750 Da, in which the cyclic structure is composed of 6.5% α-1,6 glycosidic bonds and 93.5% α-1,4 glycosidic bonds, and the average size is DP 21.

(36) Dissolving the obtained starch samples in water to prepare a solution with a concentration of 10 mg/mL; dissolving curcumin in absolute ethanol to make a solution with a mass concentration of 0.5 mg/mL; adding the curcumin solution to the main amylopectin-based cyclic glucan solution at a ratio of 10:1, placing it in a water bath at 50° C. and stirring for 3 hours at a speed of 3000 rpm, and then homogenizing at 12000 rpm for 3 minutes; placing it in an ultrasonic action device, and a treatment is performed at 10° C. for 8 minutes with a power controlled at 220 W; centrifuging, drying the obtained supernatant under vacuum to obtain a high nutritional quality of curcumin-amylopectin-based cyclic glucan inclusion.

(37) An average particle size of the obtained inclusion is 164 nm, and a loading rate of the curcumin is 2.2%; a solubility in water is 80 times higher than that of pure curcumin, and a permeability of CaCO.sub.2 cell membrane is 3.5 times higher than that of pure curcumin.

Example 7

(38) Dispersing a defatted potato starch in a solvent to obtain a 2 g/mL starch suspension, and adding an acid catalyst to perform a starch degradation reaction; after the degradation is completed, dissolving the obtained starch in a buffer, and adding a bacteria-derived trans-glycosidase preparation (sugar chain branching activity/depolymerization activity=55.0) and reacting for 12 hours to obtain an amylopectin-based cyclic glucan with an average molecular weight of 6700 Da, in which the cyclic structure is composed of 6.5% α-1,6 glycosidic bonds and 93.5% α-1,4 glycosidic bonds, and the average size is DP 21.

(39) Dissolving the obtained starch samples in water to prepare a solution with a concentration of 5 mg/mL; dissolving lycopene in absolute ethanol to make a solution with a mass concentration of 0.3 mg/mL; adding the lycopene solution to the main amylopectin-based cyclic glucan solution at a ratio of 20:1, placing it in a water bath at 40° C. and stirring for 5 hours at a speed of 2000 rpm, and then homogenizing at 10000 rpm for 5 minutes; placing it in an ultrasonic action device, and a treatment is performed at 4° C. for 2 minutes with a power controlled at 250 W; centrifuging, drying the obtained supernatant under vacuum to obtain a high nutritional quality of lycopene-amylopectin-based cyclic glucan inclusion.

(40) An average particle size of the obtained inclusion is 455 nm, and a loading rate of the lycopene is 1.7%; a solubility in water is 110 times higher than that of pure lycopene, and a permeability of CaCO.sub.2 cell membrane is 5.2 times higher than that of pure lycopene.

Example 8. Process Optimization

(41) Referring to example 7, the concentration of the amylopectin-based cyclic glucan solution is changed from 5 mg/mL to 0.1 mg/mL and 15 mg/mL, and the corresponding solution dosage is adjusted to ensure that a mass ratio of the amylopectin-based cyclic glucan to lycopene is the same, other conditions remain unchanged, and corresponding products are prepared. The results of the obtained products are shown in Table 2.

(42) TABLE-US-00002 TABLE 2 Results of products prepared with different amylopectin-based cyclic glucans. CaCO.sub.2 cell Solubility membrane Average in water permeability particle Loading (increased (increased Concen- size ratio multiples over multiples over trations (nm) (%) uninclusion) uninclusion) 0.1 mg/mL 406 1.1 115 5.2   5 mg/mL 455 1.7 110 5.2  15 mg/mL 518 0.5 109 4.7

Comparative Example 3

(43) Referring to Example 7, the amylopectin-based cyclic glucan is replaced with a β-cyclodextrin and other conditions remained unchanged to prepare a corresponding product.

(44) The solubility of the obtained product in water is increased by 1.1 times, and the CaCO.sub.2 cell membrane permeability is increased by 0.7 times.