Maltodextrin with good embedding performance and moisture resistance and preparation method thereof

Abstract

This invention provides a maltodextrin with good encapsulation performance and moisture resistance, and its preparation method. This invention belongs to the field of food processing technology. The preparation method of the maltodextrin with good encapsulation performance and moisture resistance provided by this invention includes the following steps: (1) adding 4-α-glycosyltransferase to a waxy cassava starch slurry for a first-stage enzymatic hydrolysis, inactivating the enzyme, and obtaining a waxy cassava starch slurry; (2) after heat preservation treatment of the waxy cassava starch slurry, adding a heat-resistant starch branching enzyme solution for a second-stage enzymatic hydrolysis, inactivating the enzyme, and obtaining a macrocyclic dextrin slurry; (3) centrifuging the macrocyclic dextrin slurry to remove impurities, freeze-drying, and obtaining maltodextrin with good encapsulation performance and moisture resistance. The maltodextrin prepared by the method of this invention has good encapsulation and adsorption performance, extremely high water solubility and slow digestion characteristics, and also has the advantages of low viscosity, high transparency, non-retrogradation, strong freeze-thaw stability, and good moisture resistance.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a maltodextrin with good encapsulation properties and moisture resistance, and its preparation method. Background Technology

[0002] In recent years, traditional cyclodextrins, maltodextrins, and the emerging highly branched cyclodextrins have made significant progress in food processing and functional foods. However, research shows that traditional cyclodextrins (α, β, γ-CD) have small molecular weights and limited cyclic cavity sizes, which cannot meet the requirements for flavor adsorption and encapsulation in food. Ordinary maltodextrins lack cyclic structures, have high reducing sugar content, are hygroscopic, have high water-soluble sweetness, and low transparency. Although emerging highly branched cyclodextrins have high branching, their degree of cyclization is low (below 15%), and they do not possess the unique cavity structure similar to macrocyclodextrins. The encapsulation effect of highly branched cyclodextrins on natural active substances is not ideal, which prevents many of their advantages from being reflected in some functional foods.

[0003] Macrocyclodextrin (LR-CD), also known as cyclic amylose, is a general term for cyclic dextran with a degree of polymerization greater than 9, linked by α-1,4 glycosidic bonds. Compared with common α, β, and γ-CD, LR-CD has extremely high water solubility, as well as low viscosity and non-retrogradation. Its hollow structure is hydrophilic on the outside and hydrophobic on the inside, exhibiting great flexibility and unique encapsulation and adsorption properties, capable of encapsulating some larger molecules. It has been reported that the complexation of macrocyclodextrin with phenolic compounds, resveratrol, and vitamin E can improve the stability of the substances. Research results show that the type of starch (amylose content), reaction time, reaction temperature, and the type and amount of enzyme added have a significant impact on the quality and yield of macrocyclodextrin. However, macrocyclodextrin has very few peripheral branches on its cyclic outer ring, and its molecular network structure is not dense enough, leaving considerable room for improvement in encapsulation stability and moisture resistance.

[0004] 4-α-glycosyltransferases (4αGT, EC 2.4.1.25) are typically found in the GH 13, GH 57, and GH 77 families of glycoside hydrolases. As a glycosyltransferase, 4αGT can be used to prepare macrodextrins via cyclization reactions, where one end of an α-glucan is linked to the other end via an α(1→4) glycosidic bond, producing cyclic α-glucans with a DP of 16 or higher (called macrodextrins). It has been reported that 4αGT from the thermostable bacterium *T. aquaticus* can catalyze the synthesis of LR-CD from amylose with a yield as high as 84% ​​and a minimum degree of polymerization of 22. Macrodextrins, with their larger luminal structures, are potential solubilizers for larger guest molecules.

[0005] 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) belongs to the GH13 family of glycosidic hydrolases. Using different starches as substrates, GBE catalyzes the breaking of α-1,4-glycosidic bonds and the formation of new α-1,6-glycosidic bonds, increasing the branching degree and altering the branching positions of starch molecules. This rearranges starch molecules into multi-branched cluster structures, thereby improving starch solubility, resistance to retrogradation, and slow digestibility. Therefore, highly branched starches and starch derivatives (such as highly branched cyclodextrins) prepared by GBE have wide applications in food, biomedicine, and other fields, showing great promise for industrial applications.

[0006] Waxy corn starch, with its high amylopectin content (amylose content ≤4%), serves as an excellent hydrolytic substrate for 4-α-glycosyltransferases and starch branching enzymes, resulting in thorough hydrolysis and short hydrolysis time. However, its high cost restricts the development of downstream enzymatic hydrolysis industries. Therefore, selecting the right starch type for maltodextrin preparation, ensuring core product performance indicators, and reducing costs have become key challenges. Recent research indicates that compared to waxy corn starch and waxy potato starch, waxy cassava starch has a high proportion of DP 6-12 short branches in its amylopectin structure, resulting in stronger hydrogen bonding with water. Consequently, it exhibits superior thickening properties, freeze-thaw stability, and anti-aging properties in refrigerated and frozen foods, along with higher transparency. Moreover, cassava starch is a cheaper and more readily available source. Summary of the Invention

[0007] The purpose of this invention is to overcome the technical problems of ordinary maltodextrin in the prior art, such as lack of cyclic structure, high reducing sugar content, easy moisture absorption, high water solubility and sweetness, low transparency, and unsatisfactory effect of highly branched cyclic dextrin on encapsulating natural active substances and high raw material costs.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a method for preparing maltodextrin with good encapsulation properties and moisture resistance, comprising the following steps:

[0010] (1) Add 4-α-glycosyltransferase to the waxy cassava starch slurry to carry out the first stage of enzymatic hydrolysis and inactivate the enzyme to obtain the waxy cassava starch slurry.

[0011] (2) After the waxy cassava starch liquid is kept warm, a heat-resistant starch branching enzyme solution is added to carry out the second stage of enzymatic hydrolysis and enzyme inactivation to obtain macrocyclodextrin liquid.

[0012] (3) The macrocyclic dextrin liquid is centrifuged to remove impurities and freeze-dried to obtain maltodextrin with good encapsulation performance and moisture resistance.

[0013] Preferably, the amylopectin content in the waxy cassava starch is ≥95%, and the proportion of DP 6-12 short amylopectin in the amylopectin is ≥30%; the mass concentration of the waxy cassava starch slurry is 15-40%.

[0014] Preferably, the amount of 4-α-glycosyltransferase added is 50-500 U per gram of waxy cassava starch; the pH of the first stage of enzymatic hydrolysis is 7.0-9.0, the temperature is 50-60℃, and the time is 15-30 min.

[0015] Preferably, the method for preparing the enzyme solution of the 4-α-glycosyltransferase includes the following steps:

[0016] (I) Using the 4-α-glycosyltransferase gene in Thermophilus HB8 as a template, the 4αGT gene was amplified using primers;

[0017] (II) The 4αGT gene was ligated into an expression vector to obtain the expression plasmid pET-32a(+)-4GT;

[0018] (III) The expression plasmid pET-32a(+)-4GT was transformed into Escherichia coli to obtain recombinant genetically engineered E. coli BL21 / pET-32a(+)-4GT;

[0019] (IV) The recombinant genetically engineered bacteria E.coli BL21 / pET-32a(+)-4GT was induced and cultured, and the bacterial cells were collected, broken, and the supernatant was obtained.

[0020] (V) The supernatant was freeze-dried to obtain 4-α-glycosyltransferase.

[0021] Preferably, the temperature for heat preservation in step (2) is 65-70℃ and the time is 5-10 min; the amount of heat-resistant starch branching enzyme added is 50-500 U per gram of waxy cassava starch; the pH of the second stage of enzymatic hydrolysis is 7.0-9.0, the temperature is 65-75℃, and the time is 6-12 h.

[0022] Preferably, the thermoresistant starch branching enzyme is a mutant obtained by mutating histidine at position 408 of starch branching enzyme GBE to cysteine ​​and tryptophan at position 536 to cysteine. The nucleotide sequence of the mutant is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0023] This invention provides a maltodextrin with good encapsulation properties and moisture resistance.

[0024] The present invention also provides a 4-α-glycosyltransferase.

[0025] Beneficial effects:

[0026] This invention uses high-proportion short-branched waxy cassava starch as raw material. The 4-α-glycosyltransferase obtained from the recombinant strain of Escherichia coli, which is transformed by the plasmid of thermophilic bacterium HB8, is sequentially enzymatically hydrolyzed with a molecularly modified starch branching enzyme. After centrifugation to remove impurities, the product is freeze-dried to obtain maltodextrin.

[0027] The maltodextrin prepared by the method of this invention exhibits excellent encapsulation and adsorption properties, extremely high water solubility, and slow digestibility. Furthermore, it possesses advantages such as low viscosity, high transparency, non-retrogradation, strong freeze-thaw stability, and good moisture resistance. It can be widely used as a dispersion carrier for encapsulation and adsorption in flavorings and solid beverages (instant tea powder, coffee, etc.), aligning with the current trends of natural health and clean labeling. Attached Figure Description

[0028] Figure 1 The left and right images are comparison diagrams of the hygroscopicity of the maltodextrin products prepared in Comparative Example 1 and Example 5, respectively.

[0029] Figure 2 The images show a comparison of the transparency of 30% solutions of maltodextrin products prepared in Comparative Example 1 and Example 5. The left image is the control, and the right image is Example 5. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] DP 6-12 short amylopectin refers to amylopectin with a length of 6-12.

[0032] The culture media used in the following examples are as follows:

[0033] LB medium: tryptone: 10 g / L, yeast extract: 5 g / L, NaCl: 10 g / L, agar (added when preparing LB solid plates): 20 g / L, mixed with deionized water, and autoclaved at 121°C for 20 min.

[0034] TB medium: tryptone: 12 g / L, yeast extract: 24 g / L, glycerol: 10 g / L, add deionized water and stir well, autoclave at 121℃ for 20 min.

[0035] YA medium: Yeast extract: 5g / L, add deionized water and stir well, autoclave at 121℃ for 20min.

[0036] Example 1

[0037] Preparation of 4-α-glycosyltransferase:

[0038] (I) Using the 4-α-glycosyltransferase gene template of Thermus thermophilus HB8 (GenBank accession number BAD71084) preserved in the laboratory, primers were designed. The forward primer sequence was 5′-GCCGCCATATGAGATTGGCAGGTATTTTA-3′ (as shown in SEQ ID NO: 3); the reverse primer sequence was 5′-GGCCCAAGCTTAAACTTCTCTTCCGTAAAT-3′ (as shown in SEQ ID NO: 4), with restriction sites of Nde I and HindIII, respectively. The 4αGT gene was obtained by PCR amplification.

[0039] PCR reaction system (50 μL): 33.5 μL of distilled water (sterilized), 10 μL of 5×PS Buffer, 4 μL of dNTPs, 1 μL of template, 0.5 μL each of forward and reverse primers, and 0.5 μL of DNA polymerase.

[0040] PCR reaction conditions: 94℃ for 4 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 110 s, 30 cycles, 72℃ for 10 min.

[0041] (II) The PCR product (4αGT gene) was purified and ligated into the vector pET-32a(+) to obtain the ligation product pET-32a(+)-4GT. The ligation product was transformed into E. coli DH5α competent cells and cultured overnight at 37°C on LB agar plates (containing 100 μg / mL Amp (ampicillin)). Single clones were picked and cultured in LB liquid medium (containing 100 μg / mL Amp) with shaking for 8 h. The plasmid pET-32a(+)-4GT was extracted and identified by double enzyme digestion and sequencing.

[0042] (III) After verification, transfer to E. coli BL21(DE3) and incubate on LB agar plates (containing 100 μg / mL Ampicillin) at 200 rpm for 8 h with shaking at 200 rpm in a 37°C incubator until OD. 600 =0.8, preserve the bacterial strain, and you will get the recombinant genetically engineered bacteria E.coli BL21 / pET-32a(+)-4GT.

[0043] (IV) Recombinant E. coli BL21 / pET-32a(+)-4GT was inoculated into LB medium at a 2% inoculum volume fraction and cultured at 37℃ and 200 r / min for 8 h. Then, it was transferred to TB medium at a 5% inoculum volume fraction and cultured at 37℃ and 200 r / min for 2 h. IPTG (final concentration 0.05 mmol / L) was added for induction, and the temperature was lowered to 25℃ for induction expression of recombinant protein. The peak expression level was measured every 5 hours. After 20 h of induction expression, the bacterial cells were collected by centrifugation at 8000 rpm and the supernatant was retained. The extracellular activity of the modified 4-α-glycosyltransferase was measured. The bacterial cells were suspended in 50 mmol / L Na2HPO4-citric acid buffer (pH 6.6) and sonicated at 300 W for 10 s on and 10 s off for 20 min. The cell wall was broken up by centrifugation at 8000 rpm and the supernatant was measured. The intracellular activity of the modified 4-α-glycosyltransferase was measured.

[0044] (V) The supernatant was freeze-dried for 48 hours to obtain 4-α-glycosyltransferase.

[0045] Thermophilic bacterium HB8 was inoculated into LB medium at a 2% inoculum volume fraction and cultured at 37°C and 200 rpm for 8 h. Then, it was transferred to TB medium at a 5% inoculum volume fraction and cultured at 37°C and 200 rpm for 2 h. The cells were collected by centrifugation at 8000 rpm, and the supernatant was retained. The extracellular activity of 4-α-glycosyltransferase before modification was measured. The cells were suspended in 50 mmol / L Na2HPO4-citric acid buffer (pH 6.6) and sonicated at 300 W for 10 s on and 10 s off for 20 min. The cell wall was then centrifuged at 8000 rpm to obtain the cell wall disruption supernatant. The intracellular activity of 4-α-glycosyltransferase before modification was measured.

[0046] Take 0.25 mL of substrate (0.5% soluble starch solution), 0.05 mL of 1 g / dL maltose solution, and 0.6 mL of 0.05 mol / L Na₂HPO₄ solution, respectively. 4- In a test tube, citrate buffer (pH = 6.6) was preheated at 70°C for 10 min. 0.1 mL of diluted enzyme solution (enzyme sample: buffer = 1:5) was added, the mixture was shaken to mix, and the reaction was allowed to proceed for 10 min. The reaction was then terminated by boiling in a water bath for 15 min. 10 mL of iodine solution (0.2% KI + 0.02% I₂) was added to the above reaction system, mixed, and the absorbance was measured at 620 nm.

[0047] Enzyme activity unit definition: Under the enzyme activity measurement system, the amount of enzyme required for the absorbance value to decrease by 1% per minute is defined as one enzyme activity unit.

[0048] Methods for determining enzyme activity characteristics.

[0049] Enzyme activity calculation formula:

[0050] Enzyme activity (U / mL) = (OD of control group) 660 -Experimental group OD 660 ) / Control group OD 660 / Reaction time × Dilution factor × 100

[0051] Whole cell enzyme activity is the sum of extracellular enzyme activity and intracellular enzyme activity.

[0052] Table 14. Changes in the enzyme activity characteristics of α-glycosyltransferase

[0053] name Whole cell enzyme activity (U / mg) Extracellular enzyme activity (U / mg) Intracellular enzyme activity (U / mg) Before renovation 130.12 90.65 39.47 After renovation 220.44 168.35 52.09

[0054] Therefore, the modified 4-α-glycosyltransferase provided by this invention has a 69% higher whole-cell enzyme activity and a 32% and 85.7% higher intracellular and extracellular enzyme activity, respectively. The modified 4-α-glycosyltransferase was then used in subsequent maltodextrin preparation examples 3-5.

[0055] Example 2

[0056] Starch branching enzyme derived from Aquifex aeolicus VF5 was modified using semi-rational design, targeting starch branching enzyme (GBE). After modification, histidine at position 408 was mutated to cysteine, and tryptophan at position 536 was mutated to cysteine. The nucleotide sequence of the resulting mutant is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The preparation method is as follows:

[0057] (i) Amplify the gene sequence gbe of starch branching enzyme (as shown in SEQ ID NO: 5). Using the pHT01-GBE (vector pHT01, with the gene gbe inserted into the vector via BamHI and SmaI restriction sites) constructed in the laboratory in the previous stage as a template, design primers (sequences are shown in Table 2) and perform PCR amplification using the primers below.

[0058] Table 2 Primer names and sequence names

[0059]

[0060] 1 The underlined bases correspond to the mutated amino acids.

[0061] The PCR reaction system was slightly modified from the conditions set in the STAR Primer kit instructions: 5× PrimeSTAR Buffer (Mg 2+10 μL of Plus, 1 μL of template DNA, 4 μL each of forward and reverse primers (4, 10 μM), 0.5 μL of PrimeSTAR HSDNA Polymerase (2.5 U / μL), 4 μL of dNTPs (2.5 mM each), and finally 26.5 μL of ultrapure water.

[0062] PCR amplification conditions: 98℃, 1 min 30 s; 18 cycles (98℃ 30 s, 51℃ 15 s, 72℃ 9 min 10 s); 72℃ 5 min. The purified gbe2 gene fragment was obtained after electrophoresis and recovery from the electrophoretic gel.

[0063] (ii) The purified gbe2 gene fragment was digested with DpnI enzyme, and the digestion product was purified by column chromatography. The purified product was transformed into Escherichia coli DH5α, plated on LB agar plates containing Amp (final concentration 100 μg / ml), and cultured for 16 h. Colony PCR was then performed, and the samples were sent to a company for sequencing. After successful sequencing, the positive plasmid was named pHT01-GBE2, and the starch branching enzyme mutant in it was named GBE2 (H408C-W536C).

[0064] (iii) The recombinant plasmid pHT01-GBE2 was transformed into Bacillus subtilis DB403. Single transformants were selected and cultured overnight at 37°C on LB medium supplemented with chloramphenicol (25 mg / ml). They were then transferred to TB medium and inoculated at a 2% volume ratio until the bacterial OD... 600 When the cell growth reached 0.8, IPTG (final concentration 1 mM) was added to induce expression. After culturing for another 24 hours, the culture medium was transferred to a sterile centrifuge tube and centrifuged at 8000 rpm for 10 minutes to precipitate the cells. The supernatant was discarded, and the cells were resuspended in YA medium (precipitate:YA medium = 1:5 volume ratio). Culturing continued for another 72 hours. Centrifugation at 12000 rpm for 2 minutes precipitated the cells again; the resulting supernatant was the crude enzyme solution.

[0065] (iv) The crude enzyme solution was fermented at 37°C and 200 r / min for 72 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 30 min to remove the precipitate and retain the supernatant. Ammonium sulfate was then slowly added to the supernatant at a ratio of 0.22 g / ml. The mixture was allowed to stand at 4°C for 10 min. The mixture after adding ammonium sulfate was centrifuged at 8000 rpm for 30 min to remove the supernatant. The supernatant was then redissolved with an equal volume of 20 mm Tris. The pH of the redissolved solution was adjusted to 7.5 and purified using an anion exchange column to obtain the thermostable starch branching enzyme.

[0066] The elution buffers used for purification were: 0.1M NaCl Tris-HCl (pH=7.5), 0.2M NaCl Tris-HCl (pH=7.5), 0.4M NaCl Tris-HCl (pH=7.5), 0.6M NaCl Tris-HCl (pH=7.5), 0.8M NaCl Tris-HCl (pH=7.5), 1M NaCl Tris-HCl (pH=7.5), and 2M NaCl Tris-HCl (pH=7.5).

[0067] Methods for determining enzyme activity characteristics.

[0068] Enzyme activity calculation formula:

[0069] Enzyme activity (U / mL) = (OD of control group) 660 -Experimental group OD 660 ) / Control group OD 660 / Reaction time × Dilution factor × 100

[0070] Specifically, the formula for calculating remaining enzyme activity is:

[0071] Heat half-inactivation time: Wild-type starch branching enzyme and starch branching enzyme mutant were incubated at 85°C and samples were taken at certain intervals. They were then quickly placed on ice to cool for 5 minutes. The enzyme activity of wild-type starch branching enzyme and starch branching enzyme mutant was determined according to the enzyme activity calculation formula above. The activity of the unincubated enzyme solution was defined as 100%.

[0072] Heat half-inactivation temperature: Wild-type starch branching enzyme and starch branching enzyme mutant were incubated at different temperatures (60-90℃) for 10 min and then sampled. The samples were then quickly placed on ice to cool for 5 min. The enzyme activity of wild-type starch branching enzyme and starch branching enzyme mutant was determined according to the enzyme activity calculation formula above. The activity of the unincubated enzyme solution was defined as 100%.

[0073] The wild-type amyloid branching enzyme gene was constructed from Suzhou Genewise Biotechnology Co., Ltd.

[0074] Table 3 Changes in enzyme activity characteristics

[0075] name <![CDATA[T 50 (℃)]]> <![CDATA[t 1 / 2 (min)]]> Enzyme activity (U / mg) GBE 83.52 25 22400 GBE2 87.37 75 29780

[0076] As shown in Table 2, the mutant of starch branching enzyme has a 32.9% higher enzyme activity, a 3.85℃ higher thermal half-inactivation temperature, and a 50-min longer thermal half-inactivation time. It has higher activity and significantly improved heat resistance.

[0077] Example 3

[0078] A method for preparing a maltodextrin with good encapsulation properties and moisture resistance includes the following steps:

[0079] (1) Waxy cassava starch with a branched starch content of 97%, a DP 6-12 (the length of branched starch is 6-12) short branched starch content of 40%, and good packaging without mold or insect damage was selected as raw material to prepare a waxy cassava starch slurry with a mass concentration of 15%. 4-α-glycosyltransferase (prepared in Example 1) was added at 100 U / g (waxy cassava starch). The first stage of enzymatic hydrolysis was carried out under the conditions of pH 7.5, temperature 50℃ and time 30 min. After the enzymatic hydrolysis was completed, the enzyme was inactivated (held at 121℃ for 30 min) to obtain the waxy cassava starch slurry.

[0080] (2) After cooling the waxy cassava starch solution to 70°C and keeping it warm for 10 min, add heat-resistant starch branching enzyme (prepared in Example 2) at 50 U / g (waxy cassava starch), react for 12 h at pH 8.0 and temperature 70°C, and inactivate the enzyme at 100°C for 30 min to obtain macrocyclodextrin solution.

[0081] (3) The obtained macrocyclodextrin solution was centrifuged at 5000 r / min for 5 min to remove undegraded starch impurities. The supernatant was then precipitated with 2 times its volume of anhydrous ethanol (4℃). The supernatant was then removed by centrifugation at 5000 r / min for 5 min. The solution was then placed in a fume hood for 1 h. After the alcohol had completely evaporated, 3 times the mass of the precipitate was added to redissolve the solution. The solution was then frozen at ultra-low temperature and dried in a freeze dryer. The solution was then ground in a mortar and passed through a 100-mesh sieve. The sieve residue was used to obtain the special maltodextrin.

[0082] Example 4

[0083] (1) Waxy cassava starch with a branched starch content of 95%, a DP 6-12 (the length of branched starch is 6-12) short branched starch content of 35%, and good packaging without mold or insect damage was selected as raw material to prepare a waxy cassava starch slurry with a mass concentration of 20%. 4-α-glycosyltransferase (prepared in Example 1) was added at 200 U / g (waxy cassava starch). The first stage of enzymatic hydrolysis was carried out under the conditions of pH 7.0, temperature 60℃ and time 20 min. After the enzymatic hydrolysis was completed, the enzyme was inactivated (121℃, 30 min) to obtain the waxy cassava starch slurry.

[0084] (2) After the waxy cassava starch solution is cooled to 70°C and kept warm for 10 min, a heat-resistant starch branching enzyme (prepared in Example 2) is added at 200 U / g (waxy cassava starch). The reaction is carried out at pH 9.0 and temperature 65°C for 8 h, and then inactivated at 100°C for 30 min to obtain a macrocyclodextrin solution.

[0085] (3) The obtained macrocyclodextrin solution was centrifuged at 5000 r / min for 5 min to remove undegraded starch impurities. The supernatant was then precipitated with 2 times its volume of anhydrous ethanol (4℃). The supernatant was then removed by centrifugation at 5000 r / min for 5 min. The solution was then placed in a fume hood for 1 h. After the alcohol had completely evaporated, 3 times its volume of deionized water was added to reconstitute the solution. The solution was then frozen at ultra-low temperature and dried in a freeze dryer. The solution was then ground in a mortar and passed through a 100-mesh sieve to obtain the special maltodextrin.

[0086] Example 5

[0087] (1) Waxy cassava starch with a branched starch content of 98%, a DP 6-12 (the length of branched starch is 6-12) short branched starch content of 45%, and good packaging without mold or insect damage was selected as raw material to prepare a waxy cassava starch slurry with a mass concentration of 30%. 4-α-glycosyltransferase (prepared in Example 1) was added at 200 U / g (waxy cassava starch). The first stage of enzymatic hydrolysis was carried out under the conditions of pH 9.0, temperature 55℃ and time 30 min. After the enzymatic hydrolysis was completed, the enzyme was inactivated (121℃, 30 min) to obtain the waxy cassava starch slurry.

[0088] (2) After the waxy cassava starch solution is cooled to 70°C and kept warm for 10 min, a heat-resistant starch branching enzyme (prepared in Example 2) is added at 300 U / g (waxy cassava starch). The reaction is carried out at pH 9.0 and temperature 65°C for 6 h, and then inactivated at 100°C for 30 min to obtain a macrocyclodextrin solution.

[0089] (3) The obtained macrocyclodextrin solution was centrifuged at 5000 r / min for 5 min to remove undegraded starch impurities. The supernatant was then precipitated with 2 times its volume of anhydrous ethanol (4℃). The supernatant was then removed by centrifugation at 5000 r / min for 5 min. The solution was then placed in a fume hood for 1 h. After the alcohol had completely evaporated, 3 times its volume of deionized water was added to reconstitute the solution. The solution was then frozen at ultra-low temperature and dried in a freeze dryer. The solution was then ground in a mortar and passed through a 100-mesh sieve to obtain the special maltodextrin.

[0090] Comparative Example 1

[0091] 1. Preparation of the slurry: Accurately weigh 10.0 g of corn starch (50% amylopectin content), add a small amount of water to adjust the concentration to 30%, and adjust the pH value to 6.0 with 50 mmol / L sodium hydroxide solution.

[0092] 2. Enzymatic hydrolysis reaction: Add α-amylase at a concentration of 100 U / g (corn starch), add 0.5% calcium chloride (w / w), and hydrolyze in a 75℃ constant temperature water bath for 40 min. Quickly adjust the pH value to 2.5 with 50 mmol / L hydrochloric acid solution to inactivate the enzyme (121℃, 30 min), terminate the reaction, and cool and equilibrate for two minutes.

[0093] 3. Dextrin extraction: Neutralize with 50 mmol / L sodium hydroxide solution, add water to make up to 80 mL, centrifuge at 7800 r / min for 20 min, separate the supernatant, and dry the supernatant at 50℃ for 12 h to obtain the product.

[0094] Take the finished product from Comparative Example 1 and the maltodextrin prepared in Example 5, place them in a petri dish, and let them stand for 24 hours at 90% humidity and 30°C. The results are as follows. Figure 1 As shown. By Figure 1 It can be seen that the finished product prepared in Comparative Example 1 has poor moisture resistance and obvious clumping; while Example 5 has good moisture resistance and better powder flowability.

[0095] The maltodextrin prepared in Comparative Example 1 and Example 5 were dissolved in water to form a 30% (w / v) solution, and the transparency was observed. The results are as follows: Figure 2 As shown, the finished solution prepared in Comparative Example 1 was turbid and yellowish in color; the maltodextrin prepared in this invention had no obvious color and had higher transparency.

[0096] Comparative Example 2

[0097] (1) Select ordinary corn starch (branched starch content of 50%) with intact packaging and no mold or pests as raw material, and prepare corn starch slurry with a mass volume concentration of 30% by water. Add 4-α-glycosyltransferase (prepared in Example 1) at 200 U / g (ordinary corn starch) and carry out the first stage of enzymatic hydrolysis at pH 9.0, temperature 55℃ and time 30 min. After the enzymatic hydrolysis is completed, the enzyme is inactivated (121℃, 30 min) to obtain ordinary corn starch slurry.

[0098] (2) After the ordinary corn starch solution is cooled to 70°C and kept warm for 10 min, heat-resistant starch branching enzyme (prepared in Example 2) is added at 300 U / g (waxy cassava starch). The reaction is carried out at pH 9.0 and temperature 65°C for 6 h, and then inactivated at 100°C for 30 min to obtain macrocyclodextrin solution.

[0099] (3) The obtained macrocyclodextrin solution was centrifuged at 5000 r / min for 5 min to remove undegraded starch impurities. The supernatant was then precipitated with 2 times its volume of anhydrous ethanol (4℃). The supernatant was then removed by centrifugation at 5000 r / min for 5 min. The solution was then placed in a fume hood for 1 h. After the alcohol had completely evaporated, 3 times its volume of deionized water was added to reconstitute the solution. The solution was then frozen at ultra-low temperature and dried in a freeze dryer. The solution was then ground in a mortar and passed through a 100-mesh sieve to obtain maltodextrin.

[0100] The yield and molecular weight of the maltodextrin prepared in Comparative Example 2 and Example 5 were determined, and the results are shown in Table 4:

[0101] Finished product yield = (Maltose finished product weight / Ordinary corn starch weight) × 100%

[0102] Table 4. Yield and molecular weight of maltodextrin product

[0103] name <![CDATA[M W (relative molecular mass) Finished product yield (%) Comparative Example 2 350000 68 Example 5 102000 84

[0104] Ordinary corn starch has lower hydrolysis specificity and lower product yield; on the other hand, ordinary corn starch has a lower degree of hydrolysis and an excessively large molecular weight; while the molecular weight of Example 5 is more moderate and the product yield is also higher.

[0105] As can be seen from the above embodiments, the present invention provides a method for preparing maltodextrin with good encapsulation performance and moisture resistance, belonging to the field of food processing technology. The preparation method provided by the present invention uses low-cost and stable-source cassava starch with a high proportion of short-branched (DP 6-12) waxy fibers as raw material. 4-α-glycosyltransferase is used in the first enzymatic hydrolysis stage (liquefaction stage) to carry out hydrolysis and cyclization reactions, forming a primary product with a macrocyclic dextrin molecular structure. Then, in the second enzymatic hydrolysis stage (saccharification stage), a heat-resistant starch branching enzyme is used to carry out a grafting reaction to increase the branching degree of the macrocyclic dextrin, causing the starch molecules to rearrange to generate macrocyclic dextrin with a multi-branched cluster structure. After centrifugation to remove impurities and freeze-drying, the finished maltodextrin product is obtained.

[0106] The maltodextrin prepared by this invention has two main characteristics: firstly, a cyclization degree of over 50% and a molecular weight of 50-300 KD. Its hollow structure is hydrophilic on the outside and hydrophobic on the inside, exhibiting great flexibility and unique encapsulation and adsorption properties. Secondly, the cyclic structure has dense clusters of branches on its periphery, resulting in a compact structure with extremely high water solubility and slow digestibility. Furthermore, it possesses low viscosity, high transparency, non-retrogradation, and strong freeze-thaw stability. Simultaneously, due to its low content of reducing sugars and small molecule products, it exhibits excellent moisture resistance. It can be widely used as a dispersion carrier for encapsulation and adsorption in flavorings and solid beverages (instant tea powder, coffee, etc.), aligning with the current trends of natural health and clean labeling.

[0107] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a maltodextrin with good encapsulation properties and moisture resistance, characterized in that, Includes the following steps: (1) Add 4-α-glycosyltransferase to the waxy cassava starch slurry to carry out the first stage of enzymatic hydrolysis and inactivate the enzyme to obtain the waxy cassava starch slurry. (2) After the waxy cassava starch liquid is kept warm, a heat-resistant starch branching enzyme solution is added to carry out the second stage of enzymatic hydrolysis and enzyme inactivation to obtain macrocyclodextrin liquid. (3) The macrocyclodextrin liquid is centrifuged to remove impurities and freeze-dried to obtain maltodextrin with good encapsulation performance and moisture resistance; The 4-α-glycosyltransferase was obtained from a recombinant strain of Escherichia coli obtained by transforming the 4-α-glycosyltransferase plasmid of Thermophilus HB8. The amount of 4-α-glycosyltransferase added is 50-500 U per gram of waxy cassava starch; the pH of the first stage of enzymatic hydrolysis is 7.0-9.0, the temperature is 50-60℃, and the time is 15-30 min. The thermoresistant starch branching enzyme is a mutant obtained by mutating histidine at position 408 to cysteine ​​and tryptophan at position 536 to cysteine ​​from starch branching enzyme GBE. The nucleotide sequence of the mutant is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2. The heat preservation temperature is 65~70℃, and the time is 5~10min; The amount of heat-resistant starch branching enzyme added is 50~500U per gram of waxy cassava starch; the pH of the second stage of enzymatic hydrolysis is 7.0~9.0, the temperature is 65~75℃, and the time is 6~12h.

2. The preparation method according to claim 1, characterized in that: The waxy cassava starch contains ≥95% amylopectin, and the proportion of DP 6-12 short amylopectin in the amylopectin is ≥30%; the mass concentration of the waxy cassava starch slurry is 15~40%.

3. The preparation method according to claim 1, characterized in that: The method for preparing the enzyme solution of the 4-α-glycosyltransferase includes the following steps: (I) Using the 4-α-glycosyltransferase gene in Thermophilus HB8 as a template, the 4αGT gene was amplified using primers; (II) The 4αGT gene was ligated into an expression vector to obtain the expression plasmid pET-32a(+)-4GT; (III) The expression plasmid pET-32a(+)-4GT was transformed into Escherichia coli to obtain recombinant genetically engineered E. coli BL21 / pET-32a(+)-4GT; (IV) The recombinant genetically engineered bacteria E.coli BL21 / pET-32a(+)-4GT was induced and cultured, and the bacterial cells were collected, broken, and the supernatant was obtained. (V) The supernatant was freeze-dried to obtain 4-α-glycosyltransferase.

4. A maltodextrin with good encapsulation properties and moisture resistance obtained by the preparation method according to any one of claims 1 to 3.

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