CN114317637B

CN114317637B - Preparation method and application of polygonatum sibiricum oligosaccharide

Info

Publication number: CN114317637B
Application number: CN202111649251.9A
Authority: CN
Inventors: 孙科; 孙良; 沙凤; 熊美惠
Original assignee: Suzhou Koning Polyol Co ltd
Current assignee: Suzhou Koning Polyol Co ltd
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2023-02-07
Anticipated expiration: 2041-12-29
Also published as: CN114317637A

Abstract

The invention discloses a preparation method and application of polygonatum sibiricum oligosaccharide. The polygonatum oligosaccharide is prepared by carrying out enzymolysis on polygonatum polysaccharide, wherein the glycoside hydrolase is derived from sword-like adhesion bacterium CN-02 and is GH43 and GH37 proteins. By adjusting the concentration of the polygonatum polysaccharide, the addition amount of enzyme solution, the addition proportion of GH43 and GH37, the reaction time and other factors, the polygonatum oligosaccharide with different polymerization degrees can be obtained. The method is green and efficient, and has good industrial feasibility. Meanwhile, the prepared polygonatum sibiricum oligosaccharide has good antioxidant activity and wide application prospect in the field of food.

Description

Preparation method and application of polygonatum sibiricum oligosaccharide

Technical Field

The invention belongs to the technical field of food processing, and particularly relates to a preparation method and application of polygonatum sibiricum oligosaccharide.

Background

Polygonatum sibiricum Delar. Ex Redoute belongs to Liliaceae Polygonatum, and is widely distributed in northern hemisphere temperate zone. Huang Jingshi is sweet and tasty, is a main material of many traditional Chinese medicine formulas, and has a long-standing tradition in Qinling forest regions. Rhizoma Polygonati can be used for treating weakness of spleen and stomach, asthenia, xerostomia, cough due to lung deficiency, essence and blood deficiency, internal heat, diabetes, pulmonary tuberculosis, and tinea. Researches show that the polysaccharide in the rhizoma polygonati plays a vital role in the processes.

In recent years, studies on the biological activity of oligosaccharides have been increasing compared to polysaccharides. Oligosaccharides are generally defined as carbohydrate polymers of 2-10 monosaccharide degrees of polymerization. Because of its much smaller molecular weight compared to polysaccharides, it is more readily available to animals and microorganisms, and therefore also exhibits better biological activity. More and more oligosaccharides, such as xylo-oligosaccharides, resistant dextrins, oligomannose are widely used in the field of health foods.

However, no report on the preparation method and application of polygonatum oligosaccharide with single oligosaccharide component as the main component exists at present. Therefore, the invention develops a green and efficient preparation method of polygonatum oligosaccharide, explores the application potential of polygonatum oligosaccharide in antioxidant food, and lays the foundation of tamping for high-value utilization of polygonatum polysaccharide.

Disclosure of Invention

The invention aims to provide a preparation method and application of a high-purity single-component polygonatum oligosaccharide synthesized by an enzyme method, which can be used for directly preparing the polygonatum oligosaccharide which takes a single oligosaccharide with the polymerization degree of 3, 5 or 6 as a main component by adjusting the proportional relation of enzymes.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a method for preparing rhizoma Polygonati oligosaccharide comprises using rhizoma Polygonati polysaccharide as raw material, degrading rhizoma Polygonati polysaccharide solution with glycoside hydrolase to obtain rhizoma Polygonati oligosaccharide, centrifuging, collecting supernatant, and drying to obtain rhizoma Polygonati oligosaccharide powder.

The glycoside hydrolase is GH43 protein and/or GH37 protein; the GH43 protein/GH 37 protein is derived from Campylobacter adhesion CN-02;

the amino acid sequence of the GH43 protein is shown as SEQ ID NO. 1; the amino acid sequence of the GH37 protein is shown in SEQ ID NO. 2.

A preparation method of polygonatum sibiricum oligosaccharide specifically comprises the following steps:

s1, preparing 1-20g/L solution of polygonatum polysaccharide by using a phosphate buffer solution;

s2, adding a proper amount of two glycoside hydrolase solutions, reacting the mixed solution at 30-70 ℃ for 1-8h, then boiling for 5-10min to terminate the reaction, centrifuging, collecting the supernatant, and drying to obtain the polygonatum oligosaccharide powder.

Wherein, the glycoside hydrolase is GH43 protein and GH37 protein.

Wherein the GH43 protein is derived from E.adhaerens CN-02, and the amino acid sequence is shown in SEQ ID NO. 1.

The enzyme has not been reported by the Uniprot website BLAST (https:// www.uniprot.org/BLAST /), and is a novel GH43 protein in the GH43 family.

The GH43 protease is obtained by the following method:

the GH43 gene fragment (sequence shown In SEQ ID NO. 3) was cloned using the primers GH43-F (5'-cagcaaatgggtcgcggatccATGACTGCCATGATCCGAAACC-3') and GH43-R (5'-ttgtcgacggagctcgaattcTCAACTTGCCGCAAGGCTG-3') with the Ensiferaerens CN-02 genome as a template, followed by ligation to pET-28a (+) plasmid digested by BamHI and EcoRI by In-fusion cloning to obtain a recombinant plasmid. And (3) transforming the recombinant plasmid into escherichia coli BL21 (DE 3), culturing the obtained recombinant strain, performing IPTG induced expression, homogenizing and crushing under high pressure, centrifuging at low temperature, and collecting supernatant to obtain a crude GH43 enzyme solution.

The enzymatic activity of the GH43 protein at 35 ℃ is 35.1U/mL. The definition of enzyme activity is: the amount of enzyme required to catalytically degrade 1ug of polygonatum polysaccharide per minute was defined as 1U.

The GH37 protein is derived from E.adhaerens CN-02, and the amino acid sequence is shown in SEQ ID NO. 2.

The enzyme has not been reported yet and is a novel GH37 protein in the GH37 family by the Uniprot website BLAST discovery (https:// www.uniprot.org/BLAST /).

The GH37 protease is obtained by the following method:

an GH37 gene fragment (sequence shown In SEQ ID NO. 4) was cloned using E.adhaerens CN-02 genome as a template, GH37-F (5'-cagcaaatgggtcgcggatccATGCTGTTCGACATCGATACCG-3') and GH37-R (5'-ttgtcgacggagctcgaattcTCACGGGTTGTCAGCGCT-3') primers, and then ligated to pET-28a (+) plasmid digested by BamHI and EcoRI by In-fusion cloning to obtain a recombinant plasmid. And transforming the recombinant plasmid into escherichia coli BL21 (DE 3), culturing the obtained recombinant strain, performing IPTG induced expression, homogenizing and crushing under high pressure, centrifuging at low temperature, and collecting supernatant to obtain a crude GH37 enzyme solution.

Furthermore, the enzymatic activity of the GH37 protein at 35 ℃ is 4.8U/mL. The definition of enzyme activity is: the amount of enzyme required to catalytically degrade 1ug of polygonatum polysaccharide per minute was defined as 1U.

The GH43 protease and the GH37 protease can also be synthesized according to the amino acid sequences disclosed in SEQ ID NO.1 and 2.

And obtaining the polygonatum oligosaccharide with different polymerization degrees according to different combinations of polygonatum polysaccharide concentration, enzyme solution addition amount, GH43 and GH37 addition proportion and reaction time.

Preferably, the concentration of the polygonatum polysaccharide solution is 1-10g/L.

More preferably, the concentration of the polygonatum polysaccharide solution is 2-8g/L.

The dosage ratio of the polygonatum polysaccharide to the complex enzyme is 50-10000 (w/w).

Wherein, the addition ratio (by mass) of the GH43 and the GH37 can be as follows: 1-10:1-10.

When GH43: GH37=1, w/w, the obtained product is mainly HJOPS-3;

GH43: GH37=1, and the obtained product is mainly HJOPS-5;

GH43: GH37=1, 4,w/w, the obtained product is mainly HJOPS-6;

further, in one embodiment of the present invention, when 6mL of 5g/L polygonatum polysaccharide solution and 4mL of enzyme solution (GH 43: GH37=1, 1 w/w) are contained in 10mL of hydrolysis reaction system, the reaction is carried out at 35 ℃ for 5h, and the obtained product is mainly HJOPS-3 and accounts for 89.2% of the total proportion.

In one embodiment of the invention, when a hydrolysis reaction system of 10mL contains 8mL of 5g/L rhizoma polygonati polysaccharide solution and 2mL of enzyme liquid (GH 43: GH37=1, 2, w/w), the reaction is carried out for 5h at 35 ℃, and the obtained product is mainly HJOPS-5; accounting for 91.7 percent of the total proportion.

In one embodiment of the invention, when a hydrolysis reaction system with 10mL contains 8mL of 5g/L polygonatum polysaccharide solution and 2mL of enzyme solution (GH 43: GH37=1, 4,w/w), the reaction is carried out for 5h at 35 ℃, and the obtained product is mainly HJOPS-6; accounting for 88.4 percent of the total proportion.

The GH43 protein and the GH37 protein act synergistically to hydrolyze polygonatum polysaccharides together, and the addition ratio of the GH43 protein to the GH37 protein is controlled, so that single polygonatum oligosaccharide with different components can be obtained.

Different from the prior art, the polygonatum oligosaccharide prepared by the method is mainly polygonatum oligosaccharide with single polymerization degree, and by controlling the concentration of polygonatum polysaccharide and the addition ratio of GH43 protein to GH37 protein, polygonatum oligosaccharides with different components can be obtained.

The polygonatum oligosaccharide prepared by the method is applied to antioxidant food.

The invention has the beneficial effects that:

the invention provides a green and efficient method for preparing single-component polygonatum oligosaccharide by an enzyme method, and the prepared polygonatum oligosaccharide has high purity of single oligosaccharide and good application prospect and commercial feasibility. In addition, the invention also explores the application potential of the polygonatum sibiricum oligosaccharide in antioxidant food and realizes the high-value utilization of polygonatum sibiricum polysaccharide.

Drawings

FIG. 1 is a standard curve of concentration and peak area of Polygonatum sibiricum polysaccharide;

FIG. 2 is an HPLC analysis chart of a hydrolysate of Polygonatum sibiricum polysaccharide;

FIG. 3 is an HPLC analytical chart of a hydrolysate of Polygonatum sibiricum polysaccharide;

FIG. 4 is an HPLC analysis chart of a hydrolysate of Polygonatum sibiricum polysaccharide;

FIG. 5 shows antioxidant activity of oligosaccharide from rhizoma Polygonati.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments that can be derived by one skilled in the art from the embodiments given herein are intended to be within the scope of the invention.

The GH43 protein is derived from a glycoside hydrolase family 43 (GH 43) of a Campylobacter adhesion CN-02, the amino acid sequence of the GH43 protein is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 3; the GH37 protein is derived from a glycoside hydrolase family 37 (GH 37) of Campylobacter adhesion CN-02, and has an amino acid sequence shown as SEQ ID NO.2 and a nucleotide sequence shown as SEQ ID NO. 4.

The said adhering sisal bacteria is adhering sisal bacteria (E.adhaerens) CN-02, preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.21357.

EXAMPLE 1 preparation of the hydrolases GH43 and GH37 proteins

Using E.adhaerens CN-02 genome as template, GH43 and GH37 gene fragments (nucleotide sequences are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively) were cloned with primers GH43-F (5'-cagcaaatgggtcgcggatccATGTTCTATCGTAGCGGCGTAAC-3') and GH43-R (5'-ttgtcgacggagctcgaattcTCAGGAAGCAGCCCAGAGC-3'), primers GH37-F (5'-cagcaaatgggtcgcggatccATGCTGTTCGACATCGATACCG-3') and GH37-R (5'-ttgtcgacggagctcgaattcTCACGGGTTGTCAGCGCT-3'), respectively, and then ligated to pET-28a (+) plasmid double-digested with BamH I and EcoRI by In-fusion cloning to obtain recombinant plasmids pET-28a-GH43 and pET-28 a-37. The two recombinant plasmids are respectively transformed into Escherichia coli BL21 (DE 3), and two recombinant strains are obtained. Positive transformants were selected and inoculated in LB medium (NaCl 1%, peptone 1%, yeast powder 0.5%) at 37 ℃ overnight at 200 rpm. Subsequently, the cells were inoculated in fresh LB medium at an inoculum size of 4%, cultured at 37 ℃ and 200rpm to OD ₆₀₀ Between 0.6 and 0.8, IPTG was added to a final concentration of 0.1mM for inducible expression, at which time the temperature was adjusted to 25 ℃ and fermentation was continued for 20h. In this process, the pH was controlled to 7 with ammonia, the aeration rate was set to 0.5vvm, and the dissolved oxygen was controlled to about 30% by controlling the stirring speed and the glucose feed. After the fermentation was explained, the cells were collected by centrifugation at 4000rpm for 30min and resuspended in 100mM PBS buffer pH =7. After three washes, the homogenate was broken under high pressure. Centrifuging the crushed mixed solution at 10000rpm and 4 ℃ for 20min, and collecting the supernatant, wherein the supernatant is the crude enzyme solution.

Example 2 determination of the enzymatic Activity of the hydrolases GH43 and GH37

The polygonatum polysaccharide is prepared into a solution of 5g/L by using a phosphate buffer solution with the pH of 100mM =7.0, the total hydrolysis reaction is 5mL, and the hydrolysis reaction comprises 1mL of enzyme solution (GH 43 protease solution or GH37 protease solution) and 4mL of polygonatum polysaccharide solution. The reaction temperature is 35 deg.C, the reaction time is 10min, then boiling for 5min, centrifuging, and taking the supernatant. The definition of enzyme activity is: the amount of enzyme required to catalytically degrade 1ug of polygonatum polysaccharide per minute was defined as 1U.

The detection method of the content of the polygonatum polysaccharide is carried out by HPLC. The method specifically comprises the following steps: dissolving rhizoma Polygonati polysaccharide sample (1, 2, 3, 4, 5, 6 mg) in 1mL of pure water or filtering the reaction solution supernatant with 0.22- μm membrane filter. Followed by analysis by HPLC system. The chromatographic column is Ultrahydrogel ^TM 500(7.8×300mm)、Ultrahydrogel ^TM (7.8X 300 mm), the detector is an evaporation light detector, the mobile phase is ultrapure water solution, the flow rate is 1mL/min, and the column temperature is 35 ℃.

The oligosaccharide was determined as follows: and (3) analyzing the polymerization degree of the enzymolysis oligosaccharide component by using HPLC (high performance liquid chromatography) by taking the low-polymannan with the polymerization degree of 2-6 as a standard substance. The chromatographic column is Shodex polyvinyl alcohol amino column (NH) ₂ P-50 4E), the detector is an evaporative light detector, and the mobile phase is acetonitrile/water (0-15 min:70% -60% acetonitrile; 15-25min:60% -50%; acetonitrile; 25-30min:50% -70% acetonitrile) at a flow rate of 1mL/min and a column temperature of 30 ℃.

As can be seen from the attached figure 1, the content of the polygonatum polysaccharide is analyzed by an HPLC method, and the polygonatum polysaccharide has good linear correlation. The enzymatic activity of the GH43 protein is 35.1U/mL, and the enzymatic activity of the GH37 protein is 4.8U/mL. Meanwhile, no single GH43 protein or GH37 protein hydrolysis of polygonatum polysaccharide to generate polygonatum oligosaccharide is found.

Example 3 preparation of Polygonatum sibiricum oligosaccharide based on trisaccharide

The polygonatum polysaccharide is prepared into a 5g/L solution by using 100mM phosphate buffer solution with pH =7.0, the total hydrolysis reaction is 10mL, and the total hydrolysis reaction comprises 4mL of enzyme solution (GH 43: GH37=1, w/w) and 6mL of polygonatum polysaccharide solution. The reaction temperature is 35 ℃, the reaction time is 5h, then the mixture is boiled quickly for 5min, centrifuged, and the supernatant is taken and dried to obtain the polygonatum oligosaccharide powder.

The degree of polymerization of oligosaccharides was determined as described in example 2.

As shown in figure 2, the obtained polygonatum oligosaccharide is mainly trisaccharide (HJOPS-3) and accounts for 89.2 percent of the total proportion.

Example 4 preparation of Polygonatum sibiricum oligosaccharide based on pentasaccharide

The polygonatum polysaccharide was prepared into a 5g/L solution with 100mM phosphate buffer solution with ph =7.0, and the total hydrolysis reaction was 10mL, including 2mL of enzyme solution (GH 43: GH37=1, 2,w/w) and 8mL of polygonatum polysaccharide solution. The reaction temperature is 35 ℃, the reaction time is 5h, then the mixture is boiled quickly for 5min, centrifuged, and the supernatant is taken and dried to obtain the polygonatum oligosaccharide powder.

As can be seen from figure 3, the obtained Polygonatum sibiricum oligosaccharide is mainly pentasaccharide (HJOPS-5) and accounts for 91.7% of the total proportion.

Example 5 preparation of hexasaccharide-based Polygonatum sibiricum oligosaccharide

The polygonatum polysaccharide is prepared into a 5g/L solution by using 100mM phosphate buffer solution with pH =7.0, and the total hydrolysis reaction is 10mL, and comprises 2mL of enzyme solution (GH 43: GH37=1, 4,w/w) and 8mL of polygonatum polysaccharide solution. The reaction temperature is 35 ℃, the reaction time is 5h, then the mixture is boiled quickly for 5min, centrifuged, and the supernatant is taken and dried to obtain the polygonatum oligosaccharide powder.

The degree of polymerization of oligosaccharides was determined as described in example 3.

As shown in figure 4, the obtained polygonatum oligosaccharide is mainly hexasaccharide (HJOPS-6) and accounts for 88.4 percent of the total proportion.

Example 6 measurement of antioxidant ability of Polygonatum sibiricum oligosaccharide

The polygonatum oligosaccharide obtained in the examples 3, 4 and 5 is prepared into 0.1, 0.2, 0.4, 0.8 and 1.0g/L solution by pure water respectively. The antioxidant capacity of the polygonatum oligosaccharide is determined by using a total antioxidant capacity (T-AOC) detection kit (ABTS method) purchased from Nanjing Kongshi Techniku GmbH. The experimental procedures were performed according to the kit instructions.

The results are shown in figure 5, and the polygonatum sibiricum oligosaccharides have better antioxidant capacity, and the higher the polymer is, the stronger the antioxidant capacity is.

Sequence listing

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ggcgatgaac gtccgtctct gggccgtctg tgccgtgtag aattcctgaa cgccgaaggc 180

cgtgcagtta ccccggtgct ggttctgtcc ccggaacgtc tggacgctcg tgcgtccaaa 240

cgtcagctgg ccattgaccg tcgtgtagct cacggtcgcg atcacggcga agttactttc 300

gctattgacc tggcgggcga acgcctgcac atcagcggcc gtcaggcagg cgtacgcttc 360

cacctggaag gttcccgcta cgactatgta taccgtaccc cgaacggtga agattgcctg 420

gttgctgccg ttgaaaacgt aaaattcatt ccgcgcgcga tcaccggtga tctgaaagta 480

accggcgctt ggcagcgtga ccactctgaa gacgtggacc gttccatccc gtccccgttc 540

cgtggtccgg ttccgagccg cgcgatgtct acgtccagcg ccccgtgtcg tcgtctgatc 600

ggccctgtgg gtctgactta cgttgcgtgg tctgctgaag cgctgagcga cgccaaagca 660

gaattctctg tatggcacgc tcgcgtgccg ggcggtgtgg acggccaggg tgaagctcac 720

cgtctggcgt ccgaccgcgc cagcggcttc gatctgaacg gctggctggc aaacggctgc 780

acccgtattg aggcaggcac taccccgttc cgtcgttggg ccctgcgtcc gtccatccgt 840

atttccccgt ctatttcctc tcaacagagc agcaccaccc gtaccccgcc ggcattctgc 900

cgcatcactt ctaccaccgc tacctgttcc ctgccgagcc cgtctcgccg ttgtaccgtt 960

ggccgctgcg agcgctctaa actggaccag tggctgaacg cgctgcgtcg tccgagcaac 1020

agcctggtgt acgcttctat gctgaagctg cacgctactc tgcgtacccg ttctatccag 1080

tctactaccc gttccgaagc gaccttcttt gccgaaggtg gtccggctat ctctccggat 1140

ctgccgggtg catccaaagt gttccgtatc ctggcgtgcg aggctctggc ggaactgctg 1200

gaagacgacc gcgagcgtcg tgccgtgtgg cgtcagaagg cggatgagct gcaggcgctg 1260

ctgttcgaac gcctgtggac cggcgaaacc ttcggtgcgc gtctggctgc ggacccggcc 1320

gatcgcaaaa ttcgctatga cacctacgaa ccgtcctatc cgacttccta ctcccagctg 1380

cagccggcct ctacccgtgg taaccgctcc acccagctgg tggcacgtct ggtagctggt 1440

ggcttcattc cagaccgtac ctacgctact gaatccccaa aatcctcctt ctatgaagat 1500

gacggttatt ggcgtggccc gatttgggcc ccgaccaccc tgctgctgtg ggacggcctg 1560

cgtcgtcagg gtaaaatcga gctggctcgc gaggtcgcag aaaaattttg tagcctggcg 1620

agcaaatctg gtatggctga aaactttgac gcgcgttccg gtcgcggtct gcgcgaccgt 1680

gcatttgcat ggacctccgc agtttatctg gtactggctg catctctgtc cgccgataac 1740

ccgttctaa 1749

<210> 5

<211> 43

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 5

cagcaaatgg gtcgcggatc catgactgcc atgatccgaa acc 43

<210> 6

<211> 40

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 6

ttgtcgacgg agctcgaatt ctcaacttgc cgcaaggctg 40

<210> 7

<211> 43

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 7

cagcaaatgg gtcgcggatc catgctgttc gacatcgata ccg 43

<210> 8

<211> 39

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 8

ttgtcgacgg agctcgaatt ctcacgggtt gtcagcgct 39

Claims

1. A preparation method of polygonatum oligosaccharide is characterized in that polygonatum polysaccharide is used as a raw material, and a solution of polygonatum polysaccharide is degraded by glycoside hydrolase to obtain polygonatum oligosaccharide, wherein the glycoside hydrolase is GH43 protein and GH37 protein; the amino acid sequence of the GH43 protein is shown as SEQ ID NO. 1; the amino acid sequence of the GH37 protein is shown in SEQ ID NO. 2.

2. The method of claim 1, wherein the solution of polygonatum polysaccharides is a solution prepared from phosphate buffer solution at a concentration of 1-20 g/L.

3. The method for preparing polygonatum sibiricum oligosaccharide according to claim 1, wherein the degradation condition is a reaction at 30-70 ℃ of 1-8 h.

4. The preparation method of polygonatum oligosaccharide according to claim 1, wherein the glycoside hydrolase is GH43 protein and GH37 protein, and the addition ratio of GH43 to GH37 is 1-10 by mass.

5. The method for preparing polygonatum oligosaccharide according to claim 1, wherein the method comprises the following steps: the glycoside hydrolase GH43 protein and the GH37 protein are derived from the sword-sticking fungus CN-02, the sword-sticking fungus CN-02 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC NO 21357.

6. The method for preparing polygonatum oligosaccharide according to claim 1, wherein the polygonatum polysaccharide and glycoside hydrolase are used in a ratio of 50-10000 w/w.

7. The method for preparing polygonatum oligosaccharide according to claim 4, wherein the method comprises the following steps: the addition ratio of GH43 and GH37 is 1:1 by mass, and the polygonatum oligosaccharide mainly containing trisaccharides is prepared.

8. The enzymatic preparation method of polygonatum sibiricum oligosaccharide according to claim 4, wherein the addition ratio of GH43 and GH37 is 1:2 by mass, and polygonatum sibiricum oligosaccharide mainly containing pentasaccharide is prepared.

9. The enzymatic preparation method of polygonatum sibiricum oligosaccharide according to claim 4, wherein the addition ratio of GH43 and GH37 is 1:4 by mass, and polygonatum sibiricum oligosaccharide mainly containing hexasaccharide is prepared.

10. Use of the polygonatum oligosaccharide prepared by the preparation method according to the claims 7-9 in preparing antioxidant food.