Dendrobium huoshanense polysaccharide high-efficiency enzymolysis and oligosaccharide detection method

Abstract

This invention relates to the field of plant polysaccharide processing technology, and discloses a highly efficient enzymatic hydrolysis and oligosaccharide detection method for Dendrobium huoshanense polysaccharides. The method involves steps such as drying, impurity removal, pulverization, ethanol defatting, hot water extraction, vacuum concentration, alcohol precipitation, and Sevag deproteinization to obtain refined polysaccharides. These are then subjected to enzymatic hydrolysis. Factors such as hydrolysis time, enzyme dosage, and number of hydrolysis cycles are investigated, and the process is optimized using response surface methodology, significantly increasing the yield of Dendrobium huoshanense oligosaccharides to over 90%. Finally, HPLC-ELSD is used to detect the oligosaccharide composition. This method is highly efficient, simple, and pollution-free, providing high-quality raw materials for the development of Dendrobium huoshanense oligosaccharides and significantly increasing its extraction efficiency and economic value.

Description

Technical Field

[0001] This invention relates to the field of plant polysaccharide processing technology, and more specifically, to a method for efficient enzymatic hydrolysis of Dendrobium huoshanense polysaccharides and detection of oligosaccharides. Background Technology

[0002] Dendrobium huoshanense is a precious traditional Chinese medicine with rich medicinal value and health benefits. In recent years, with the deepening research on the active ingredients of traditional Chinese medicine, the oligosaccharide components in Dendrobium huoshanense have gradually attracted the attention of researchers. Oligosaccharides, also known as oligosaccharides, are low-density polymers composed of 2-10 monosaccharides linked by glycosidic bonds. Compared with monosaccharides and polysaccharides, oligosaccharides have unique biological activities and physiological functions. Polysaccharides, as large molecular compounds, have low absorption and utilization efficiency in the human body. As a degradation product of Dendrobium huoshanense, oligosaccharides retain some biological activity and have advantages such as good water solubility and easy absorption, showing broad application prospects in many fields. Therefore, the conversion of polysaccharides into oligosaccharides has become a research hotspot.

[0003] However, existing enzymatic hydrolysis techniques for Dendrobium huoshanense polysaccharides have many shortcomings. Differences in process conditions such as enzyme type, enzyme concentration, reaction temperature, and reaction time directly lead to changes in the composition of the hydrolysis products, resulting in oligosaccharides or monosaccharides with different molecular weight distributions. Simultaneously, variations in the degree of hydrolysis can alter the molecular weight and structural characteristics of the products, thus affecting their solubility, stability, and bioactivity. The composition and degree of polymerization of oligosaccharides are difficult to control, and the degree of degradation is not precise. Furthermore, existing methods for detecting Dendrobium huoshanense oligosaccharides suffer from low sensitivity, long analysis times, and complex operations, and lack standardized detection methods. Therefore, developing efficient enzymatic hydrolysis techniques for Dendrobium huoshanense polysaccharides and accurate oligosaccharide detection methods is of significant practical importance. This invention aims to provide relevant methods to improve hydrolysis efficiency, obtain specific oligosaccharides, and establish rapid, accurate, and sensitive detection methods, providing technical support for the quality control and application development of Dendrobium huoshanense oligosaccharides. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for efficient enzymatic hydrolysis of polysaccharides from Dendrobium huoshanense and detection of oligosaccharides, comprising:

[0005] Step 1: Dry, remove impurities, and pulverize the Dendrobium huoshanense material;

[0006] Step 2: Remove lipid-soluble small molecules using ethanol extraction;

[0007] Step 3: Obtain crude polysaccharides from Dendrobium huoshanense using hot water extraction;

[0008] Step 4: The crude polysaccharide is concentrated under reduced pressure and precipitated with alcohol, and the protein is removed using the Sevag method to obtain refined polysaccharide;

[0009] Step 5: Perform enzymatic hydrolysis on the refined polysaccharide for 5-15 hours. The enzyme dosage is 0.2 U / mL-0.6 U / mL per gram of refined polysaccharide. The hydrolysis is repeated 1-3 times. The maximum yield of oligosaccharides obtained after polysaccharide hydrolysis is greater than 90%.

[0010] Step 6: Oligosaccharide composition was determined using HPLC-ELSD.

[0011] Preferably, in step S5, β-mannanase is selected for enzymatic hydrolysis.

[0012] Preferably, in step 5, the enzymatic hydrolysis time is 10 hours.

[0013] Preferably, in step 5, the amount of enzyme used is 0.4 U / mL of enzyme added per gram of refined polysaccharide.

[0014] Preferably, in step 5, the enzymatic hydrolysis is performed twice.

[0015] Preferably, in step 5, the composition of the oligosaccharides after enzymatic hydrolysis is analyzed using the detection results of a control standard. For example... Figure 3 The HPLC-ELSD standard chromatogram of nine oligosaccharides shows that the retention time of each chromatographic peak of the oligosaccharide standard is positively correlated with its molecular weight, and the retention time from shortest to longest corresponds to the increasing order from monosaccharide to nonasugar.

[0016] Preferably, the step involves using ethanol extraction to remove fat-soluble small molecules. Specifically, the pulverized Dendrobium huoshanense material is mixed with ethanol at a certain ratio (70%-100% ethanol concentration), the extraction temperature is 50℃-80℃, and the extraction time is 1-3 hours. This step effectively removes fat-soluble impurities from Dendrobium huoshanense and improves the purity of polysaccharides.

[0017] The preferred method is to use an ethanol concentration of 85%, an extraction temperature of 65°C, and an extraction time of 2 hours.

[0018] Preferably, the crude polysaccharide of Dendrobium huoshanense is obtained by hot water extraction. The Dendrobium huoshanense material after ethanol extraction is mixed with hot water and extracted to obtain an extract containing Dendrobium huoshanense polysaccharide.

[0019] Preferably, the crude polysaccharide extract is concentrated under reduced pressure to remove excess water and increase the polysaccharide concentration. Subsequently, an appropriate amount of ethanol is added for alcohol precipitation, causing the polysaccharide to precipitate and further improving the purity of the polysaccharide.

[0020] Preferably, the Sevag method is used in the above steps to remove protein impurities from the polysaccharide. The polysaccharide precipitate after alcohol precipitation is mixed with Sevag reagent in a certain proportion, and protein impurities are removed through multiple extractions to finally obtain purified polysaccharide.

[0021] Preferably, the step uses an Ultirnate UHPLC Amide column, which has good separation performance and can effectively separate different components in Dendrobium huoshanense oligosaccharides;

[0022] Acetonitrile was used as mobile phase A, and water was used as mobile phase B. Elution was performed using 75% A isocratic elution for 20 min. This combination of mobile phases and elution method enabled the oligosaccharide components to be well separated in a short time.

[0023] The flow rate is set to 0.250 mL / min, which ensures the separation effect and detection sensitivity of the chromatographic column.

[0024] The column temperature is controlled at 30℃. A suitable column temperature helps to improve the separation efficiency and stability of the chromatographic column.

[0025] The injection volume is 5 μL. An appropriate injection volume can ensure the accuracy and repeatability of the detection results.

[0026] An Agilent 1290 ELSD detector was used, with parameters set to gain 10, gas pressure 172.37 kPa, drift tube temperature 60°C, and Nebheater temperature 60°C. The ELSD detector exhibits high sensitivity and good linear range for oligosaccharides, enabling accurate detection of oligosaccharide content and composition.

[0027] The beneficial effects of this invention are as follows: By optimizing the enzymatic hydrolysis process conditions (such as enzyme type, enzyme concentration, reaction temperature, and reaction time), this invention achieves efficient and precise degradation of Dendrobium huoshanense polysaccharides, stably obtaining oligosaccharide products with specific molecular weight distribution and degree of polymerization. This effectively solves the problems of difficulty in controlling oligosaccharide composition and degree of polymerization and inaccurate degradation degree in existing technologies, improving the stability and bioactivity of oligosaccharide products. Simultaneously, the oligosaccharide detection method provided by this invention has advantages such as high sensitivity, fast analysis speed, and simple operation, overcoming the shortcomings of existing detection methods such as low sensitivity, long analysis time, and complex operation. It establishes a standardized oligosaccharide detection method, providing reliable technical support for the quality control of Dendrobium huoshanense oligosaccharides. Furthermore, the water-soluble and easily absorbed Dendrobium huoshanense oligosaccharides obtained by this invention retain the original bioactivity of Dendrobium huoshanense, have broad application prospects, and can be widely used in multiple fields such as medicine, health products, and food, promoting the in-depth development and efficient utilization of Dendrobium huoshanense resources. Therefore, this invention has significant beneficial effects in improving the enzymatic hydrolysis efficiency of Dendrobium huoshanense polysaccharides, accurately controlling oligosaccharide products, establishing an efficient and sensitive detection method, and promoting the industrial application of Dendrobium huoshanense oligosaccharides. Attached Figure Description

[0028] Figure 1 This is a flowchart of the method of the present invention;

[0029] Figure 2 This is a liquid chromatogram of a Dendrobium sample after enzymatic hydrolysis;

[0030] Figure 3 This is a chromatogram of a mixed standard for detecting nine oligosaccharides using HPLC-ELSD. Detailed Implementation

[0031] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0032] Example 1

[0033] This embodiment proposes a method for efficient enzymatic hydrolysis of polysaccharides from Dendrobium huoshanense and detection of oligosaccharides, including:

[0034] Step 1: Dry, remove impurities and crush the Dendrobium officinale material.

[0035] Step 2: Remove fat-soluble small molecules by ethanol extraction; mix the pulverized Dendrobium huoshanense material with ethanol in a certain proportion, the concentration of ethanol is 85%, the extraction temperature is 65℃, and the extraction time is 2 hours.

[0036] Step 3: Obtain crude polysaccharides from Dendrobium huoshanense using hot water extraction; mix Dendrobium huoshanense material that has been extracted with ethanol with hot water and perform hot water extraction to obtain an extract containing Dendrobium huoshanense polysaccharides.

[0037] Step 4: The crude polysaccharide extract is concentrated under reduced pressure to remove excess water and increase the polysaccharide concentration. Then, an appropriate amount of ethanol is added for alcohol precipitation to precipitate the polysaccharide, further improving its purity. Proteins are removed using the Sevag method. The precipitated polysaccharide is then mixed with Sevag reagent in a specific ratio and extracted multiple times to remove protein impurities, ultimately obtaining purified polysaccharide.

[0038] Step 5: Perform enzymatic hydrolysis on the refined polysaccharide. Select β-mannanase for enzymatic hydrolysis. The hydrolysis time is 10 hours. The amount of enzyme added is 0.4 U / mL per gram of refined polysaccharide. The number of hydrolysis cycles is 2. The maximum yield of oligosaccharides obtained after polysaccharide hydrolysis is greater than 90%.

[0039] Step 6: Oligosaccharide composition was determined using HPLC-ELSD;

[0040] An Ultirnate UHPLC Amide column was used with acetonitrile as mobile phase A and water as mobile phase B. Elution was performed isocratically with 75% A for 20 min at a rate of 0.250 mL / min. The column temperature was controlled at 30℃, and the injection volume was 5 μL. An Agilent 1290 ELSD detector was used with the following parameters: gain 10, gas pressure 172.37 kPa, drift tube temperature 60℃, and Nebheater temperature 60℃.

[0041] Example 2

[0042] The difference between this embodiment and Embodiment 1 is that:

[0043] In step 2: the pulverized Dendrobium huoshanense material is mixed with ethanol in a certain proportion, the concentration of ethanol is 70%, the extraction temperature is 50℃, and the extraction time is 1 hour.

[0044] In step 5: the enzymatic hydrolysis time is 5 hours, the amount of enzyme used is 0.2 U / mL per gram of refined polysaccharide, and the enzymatic hydrolysis is performed once.

[0045] Example 3

[0046] The difference between this embodiment and Embodiment 1 is that:

[0047] In step 2: the pulverized Dendrobium huoshanense material is mixed with ethanol in a certain proportion, the concentration of ethanol is 100%, the extraction temperature is 80℃, and the extraction time is 3 hours.

[0048] In step 5: the enzymatic hydrolysis time is 5 hours, the amount of enzyme used is 0.6 U / mL per gram of refined polysaccharide, and the enzymatic hydrolysis is performed 3 times.

[0049] Example 4

[0050] This embodiment provides an efficient method for the preparation and detection of oligosaccharides from Dendrobium huoshanense.

[0051] First, the Dendrobium huoshanense material was dried, impurity removed, pulverized, and sieved to obtain uniform granular powder. Next, the powder was weighed and placed in a round-bottom flask, and 80% ethanol solution was added. Extraction was performed in an 80℃ water bath for 1 hour, followed by filtration and repeated extraction twice to remove lipid-soluble small molecule impurities. The residue was evaporated to dryness, and double-distilled water was added for extraction in a 90℃ water bath for 2 hours, repeated twice. The filtrates were then combined. The mixture was then concentrated under reduced pressure until no water remained, and anhydrous ethanol was added to adjust the concentration to 76%. The mixture was frozen at 4℃ for 12 hours and centrifuged at 4000 rpm for 20 minutes to obtain crude polysaccharide precipitate. After the precipitate dissolved, a Sevag mixture was added at a volume ratio of 4:1, shaken, and centrifuged or separated to remove proteins, preparing a purified polysaccharide solution, which was then freeze-dried. A 0.1 mg / mL solution of the purified polysaccharide was prepared, and β-mannanase was added. Enzymatic hydrolysis was performed at 37℃ for several hours, with optimization of the enzyme dosage, hydrolysis time, and number of hydrolysis cycles. The oligosaccharide solution was obtained by centrifugation using 3000 Da ultrafiltration centrifuge tubes to remove incompletely enzymatically hydrolyzed residual polysaccharides, and then lyophilized. Finally, HPLC-ELSD was used to analyze the oligosaccharide samples from *Dendrobium huoshanense*. The results showed that this invention significantly improved the extraction efficiency of polysaccharides from *Dendrobium huoshanense*, with an oligosaccharide yield exceeding 90% after optimization of the enzymatic hydrolysis process. Furthermore, the oligosaccharides were mainly composed of disaccharides to heptasaccharides, with a relative content of 13.51%-26.77%. This method is efficient, simple, and environmentally friendly, possessing broad application prospects and significant economic value.

[0052] Example 5

[0053] 1. Preparation of Dendrobium huoshanense powder

[0054] The Dendrobium officinale material from Huoshan was dried and impurities were removed. Then it was pulverized, passed through a 10-mesh sieve, and the uniform Dendrobium officinale powder was collected.

[0055] 2. Removal of fat-soluble small molecule components

[0056] Weigh 150g of Dendrobium powder and place it in a round-bottom flask. Add 1500mL of 80% ethanol solution at a material-to-liquid ratio of 1:10 for extraction. For the first extraction, add 1500mL of 80% ethanol solution and extract in an 85℃ water bath for 1 hour, then filter. Add another 1500mL of 80% ethanol solution to the residue from the first extraction, extract in an 80℃ water bath for 1 hour, and then filter.

[0057] 3. Extraction of crude polysaccharides from Dendrobium huoshanense

[0058] The remaining ethanol solution in the round-bottom flask was evaporated, and 1500 mL of double-distilled water was added. The mixture was extracted in a 95℃ water bath for 4 hours and the liquid was filtered out. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator until no obvious water was evaporated. Then, anhydrous ethanol was added to the concentrate to adjust the ethanol concentration to 76%. The concentrate was then frozen and stored at 4℃. After standing for 12 hours, the mixture was centrifuged at 5000 rpm for 20 minutes to obtain the crude polysaccharide precipitate of Dendrobium huoshanense.

[0059] 4. Preparation of purified polysaccharide solution

[0060] The crude polysaccharide precipitate of Dendrobium huoshanense was dissolved in a small amount of double-distilled water. The Sevag method (a mixed solution of chloroform and n-butanol) was used to mix the polysaccharide solution at a volume ratio of 4:1 and shaken to denature and precipitate the protein. The precipitated protein was removed by centrifugation or separation to obtain a purified polysaccharide solution with the protein removed.

[0061] 5. Preparation of refined polysaccharide powder

[0062] The purified polysaccharide solution after removing proteins is freeze-dried to obtain purified polysaccharide powder.

[0063] 6. Preparation of refined polysaccharide solution from Dendrobium huoshanense

[0064] Weigh 20 mg of refined polysaccharide and add it to double-distilled water to prepare a Dendrobium officinale refined polysaccharide solution with a concentration of 0.2 mg / mL.

[0065] 7. Preparation and optimization of oligosaccharides from Dendrobium huoshanense

[0066] Take 10 mL of purified polysaccharide solution and add an appropriate amount of β-mannanase solution. Enzymatically hydrolyze at 37℃ for 10 h. Select an ultrafiltration centrifuge tube with a molecular weight of 3000 Da for centrifugation to remove residual polysaccharide components that have not been completely hydrolyzed. Freeze-dry the obtained oligosaccharide solution to obtain Dendrobium huoshanense oligosaccharides.

[0067] 8. Detection of oligosaccharides from Dendrobium huoshanense

[0068] Standards for sugars 1-9 were prepared by calculating the mass ratio of each oligosaccharide according to their peak heights to obtain a mixed standard for the nine oligosaccharides. The individual standard masses were as follows: glucose (1.25 mg), sucrose (1.37 mg), raffinose (1.32 mg), fructotetrasaccharide (1.08 mg), fructopentose (1.34 mg), fructohexasaccharide (1.51 mg), fructoheptasaccharide (2.25 mg), fructooctasaccharide (2.84 mg), and fructononose (3.49 mg). The mixed standard was then analyzed by liquid chromatography. Chromatographic conditions: An Ultirnate UHPLC Amide column was used with acetonitrile as mobile phase A and water as mobile phase B, eluted isocratically with 75% A for 20 min. The flow rate was 0.250 mL / min, the column temperature was 30 ℃, the injection volume was 5 μL, and an Agilent 1290 ELSD detector was used with the following parameters set: gain 10, gas pressure 172.37 kPa, drift tube temperature 60 ℃, and Nebheater temperature 60 ℃. The detection results are as follows: Figure 3 The HPLC-ELSD standard chromatogram of nine oligosaccharides is shown. The analysis results indicate a significant positive correlation between the retention time and molecular weight of each oligosaccharide component: that is, as the number of monosaccharide units in the oligosaccharide molecule increases, its molecular weight increases, and the corresponding retention time also prolongs. Therefore, the elution order of the chromatographic peaks, from shortest to longest retention time, corresponds sequentially to monosaccharides, disaccharides, trisaccharides… up to nonasaccharides.

[0069] (1) Examination of linear relationships

[0070] Accurately pipette the mixed standard solution (concentration 0.1 mg / mL) and serially dilute it by half with 70% acetonitrile solution (extraction solvent) to obtain 10 series concentrations for injection and determination. Perform linear regression with peak area (Y) as the ordinate and concentration (X, mg / mL) as the abscissa. See the table below:

[0071]

[0072] (2) Precision test

[0073] Accurately pipette an appropriate amount of the mixed standard solution, inject it continuously for 6 measurements, record the peak area, and calculate the RSD value. Results showing an RSD value less than 3% indicate good instrument precision.

[0074] (3) Stability test

[0075] Oligosaccharide samples from Dendrobium were injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 h, and the peak areas were recorded and RSD values ​​were calculated. The results showed that the RSD value was less than 3%, indicating that the test solution had good stability within 24 h.

[0076] (4) Reproducibility test

[0077] Three parallel solutions of Dendrobium oligosaccharide were prepared from the same batch of Dendrobium sample powder and injected for analysis. The peak areas were recorded, and the RSD values ​​were calculated. The results showed that the RSD value was less than 3%, indicating that the method had good repeatability.

[0078] (5) Recovery rate experiment

[0079] Accurately weigh 6 portions of Dendrobium powder with known content, approximately 1.0 g per portion. Accurately add an appropriate amount of mixed standard solution, extract under the same extraction conditions, prepare samples, inject and determine the chromatographic peak area, and calculate the recovery rate (see [link to sample preparation]). Figure 2 The recoveries of oligosaccharides were found to be in the range of 90.2%–105.7%, with RSDs ranging from 2.1% to 2.9%, indicating that the method has good recovery rates.

[0080] 9. To optimize the enzymatic hydrolysis process of Dendrobium huoshanense polysaccharides and improve the yield of oligosaccharides, a response surface methodology (RSM) optimization scheme was used. This scheme considers three main factors: enzyme dosage, hydrolysis time, and number of hydrolysis cycles. The specific RSM design is as follows:

[0081] Select three factors and their level ranges: enzyme dosage (X1): 0.2 U / mL to 0.6 U / mL of enzyme per gram of refined polysaccharide; hydrolysis time (X2): 5 hours to 15 hours; number of hydrolysis cycles (X3): 1 to 3 times.

[0082] Response surface methodology was optimized using a central composite design (CCD). The CCD design includes combinations of factor levels, including center points, pivot points, and corner points. The specific experimental design matrix is ​​shown in the table below:

[0083]

[0084] The response variable was the yield of oligosaccharides, which was determined by HPLC-ELSD.

[0085] The data were subjected to multivariate fitting analysis using Design Expert 10.0 software, resulting in a quadratic multinomial regression equation with the oligosaccharide hydrolysis yield Y as the response value against the independent variables (enzyme dosage (X1), hydrolysis time (X2), and number of hydrolysis cycles (X3)).

[0086] Y=-6.18+95.73X1+4.31X2-0.44X3+16.07X1X2+55.52X1X3+3.82X2X3-369.71 X 2 1-0.85 X 2 2-10.65 X 23

[0087] From the significance test of the ANOVA of the regression model in Table 3, the effects of the linear terms (X1, X3), the quadratic terms, and the interaction term of the quadratic terms on the polysaccharide extraction yield were highly significant (P < 0.1), while the single enzymatic hydrolysis time had no significant effect on the results (P > 0.1). The p-value of the model was 0.001, indicating that the regression was extremely significant. The influence of each factor on the polysaccharide extraction yield was in the following order: number of hydrolysis cycles (X3) > enzyme dosage (X1) > hydrolysis time (X2).

[0088]

[0089] Analysis using DesignExpert 10.0 software yielded the following optimized enzymatic hydrolysis conditions for the polysaccharides: enzyme dosage 0.6 U / mL, hydrolysis time 10 h, and hydrolysis cycles 3 times. Under these conditions, the theoretical hydrolysis yield of Dendrobium officinale polysaccharides reached 90.05%. Based on actual conditions, repeating the experiment 3 times under these conditions resulted in a polysaccharide extraction yield of 89.92%, which is close to the predicted value. Therefore, these process parameters can be used to guide the preparation of polysaccharides and oligosaccharides from Dendrobium officinale.

[0090] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for efficient enzymatic hydrolysis of polysaccharides from Dendrobium huoshanense and detection of oligosaccharides, characterized in that, include: Step 1: Dry, remove impurities, and pulverize the Dendrobium huoshanense material; Step 2: Remove lipid-soluble small molecules using ethanol extraction; Step 3: Obtain crude polysaccharides from Dendrobium huoshanense using hot water extraction; Step 4: The crude polysaccharide is concentrated under reduced pressure and precipitated with alcohol, and the protein is removed using the Sevag method to obtain refined polysaccharide; Step 5: Perform enzymatic hydrolysis on the refined polysaccharide for 5-15 hours. The amount of enzyme used is 0.2 U / mL-0.6 U / mL per gram of refined polysaccharide. The hydrolysis is performed 1-3 times. The maximum yield of oligosaccharides obtained after polysaccharide hydrolysis is greater than 90%. β-Mannanase is selected for hydrolysis. Step 6: The oligosaccharide composition was determined by HPLC-ELSD. The oligosaccharides consisted of disaccharides to heptasaccharides, with a relative content of 13.51%-26.77%.

2. The method for enzymatic hydrolysis of polysaccharides and detection of oligosaccharides from Dendrobium huoshanense according to claim 1, characterized in that, In step S5, the enzymatic hydrolysis time is 10 hours.

3. The method for enzymatic hydrolysis of polysaccharides and detection of oligosaccharides from Dendrobium huoshanense according to claim 1, characterized in that, In step S5, the amount of enzyme used is 0.4 U / mL per gram of refined polysaccharide.

4. The method for enzymatic hydrolysis of polysaccharides and detection of oligosaccharides from Dendrobium huoshanense according to claim 1, characterized in that, In step S5, the enzymatic hydrolysis is performed twice.

5. The method for enzymatic hydrolysis of polysaccharides and detection of oligosaccharides from Dendrobium huoshanense according to claim 1, characterized in that, In step 2, during ethanol extraction, the ethanol concentration is 70%-100%, the extraction temperature is 50℃-80℃, and the extraction time is 1 hour-3 hours.

6. The method for enzymatic hydrolysis of polysaccharides and detection of oligosaccharides in Dendrobium huoshanense according to claim 4, characterized in that, The concentration of ethanol is 85%.

7. The method for enzymatic hydrolysis of polysaccharides and detection of oligosaccharides from Dendrobium huoshanense according to claim 4, characterized in that, The extraction temperature is 65℃.

8. The method for enzymatic hydrolysis of polysaccharides and detection of oligosaccharides from Dendrobium huoshanense according to claim 4, characterized in that, The extraction time is 2 hours.

9. The method for enzymatic hydrolysis of polysaccharides and detection of oligosaccharides from Dendrobium huoshanense according to claim 1, characterized in that, In step 6, an Ultirnate UHPLC Amide column was used with acetonitrile as mobile phase A and water as mobile phase B. Elution was performed isocratically with 75% A for 20 min at a flow rate of 0.250 mL / min, a column temperature of 30 ℃, and an injection volume of 5 μL. An Agilent 1290 ELSD detector was used with parameters set to gain 10, gas pressure 172.37 kPa, drift tube temperature of 60 ℃, and Nebheater temperature of 60 ℃.

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