A high-efficiency enzymatic method for releasing polyphenols from polygonatum

Abstract

The application discloses an enzymolysis method for efficiently releasing phenolic substances of rhizoma polygonati, and belongs to the field of biological enzymatic method research of rhizoma polygonati components. The rhizoma polygonati enzymolysis method comprises the following steps: crushing and screening rhizoma polygonati, weighing rhizoma polygonati powder and a composite enzyme composed of papain, beta-glucanase and xylanase, adding the composite enzyme into water, adjusting a pH value, activating in a constant-temperature water bath kettle first, then performing an enzymolysis reaction, taking out immediately after the enzymolysis is completed, inactivating, and obtaining an enzymolysis product; then adding ethanol into the enzymolysis product in a flask, performing a boiling water bath extraction, filtering while hot, obtaining an extract, performing rotary evaporation concentration, obtaining an alcohol extract, placing the precipitate in a flask, adding pure water, performing a boiling water bath extraction, filtering while hot, obtaining an extract, and obtaining a water extract, and the method ends; the method can make the phenolic components in rhizoma polygonati extraction obviously change, a plurality of new substances appear, and the new substances have good antioxidant capacity, and the method provides a scientific basis for the field of rhizoma polygonati deep processing technology.

Description

Technical Field

[0001] This invention relates to an enzymatic hydrolysis method for efficiently releasing phenolic substances from Polygonatum, belonging to the field of bioenzymatic research on Polygonatum components. Background Technology

[0002] Polygonati Rhizoma is a herbaceous plant belonging to the genus Polygonati. It has a long history of being used both as food and medicine. Ancient texts such as the *Mingyi Bielu* (Records of Famous Physicians) officially name Polygonati Rhizoma as "primarily tonifying the middle energizer and replenishing qi, dispelling wind and dampness, and calming the five internal organs. Long-term consumption can lighten the body, prolong life, and eliminate hunger." The *Shiliao Bencao* (Dietary Therapy Materia Medica) records that "consuming Polygonati Rhizoma can prevent aging and hunger." The *Chinese Pharmacopoeia* defines it as "tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys." Modern pharmacological research shows that plants in the genus Polygonati mainly contain functional components such as steroidal saponins, polysaccharides, and phenols.

[0003] Currently, the effective utilization of Polygonatum components, besides processing it to achieve full component intake, also involves adding Polygonatum extract to health food production to achieve product diversification. The extraction of Polygonatum components typically employs the methods mentioned in the 2020 edition of the Chinese Pharmacopoeia: first, small molecules such as total phenols and total saponins are extracted using alcohol, followed by water extraction of large molecules such as polysaccharides and proteins. However, due to the incomplete disruption of the Polygonatum cell wall and the different forms of active ingredients within plant cells, the traditional alcohol-water extraction process cannot fully extract the effective components from Polygonatum.

[0004] Enzymatic methods are widely used in the food industry and are a relatively low-cost method. They can degrade cell walls, which can eliminate physical barriers, open cells, and disrupt the interaction between active ingredients and cell wall structure, thus facilitating the extraction of active ingredients. However, there are currently no reports on the effective extraction of phenolic substances from Polygonatum sibiricum. Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, this invention provides an efficient enzymatic hydrolysis method for releasing phenolic substances from Polygonatum sibiricum. This method first treats Polygonatum sibiricum powder with a compound enzyme, followed by alcohol extraction and water extraction, which effectively promotes the release of phenolic substances from Polygonatum sibiricum, increases the amount of new phenolic substances, and thus also increases the content of total saponins and total phenols in Polygonatum sibiricum extract.

[0006] The purpose of this invention is to provide an efficient enzymatic hydrolysis method for releasing polysaccharide substances from styrax, the method comprising the following steps:

[0007] (1) Powdered and sieved Polygonatum, weighed Polygonatum powder and a complex enzyme composed of papain, β-glucanase and xylanase, added to water, and adjusted the pH value. Activated in a constant temperature water bath first, and then carried out the enzymatic hydrolysis reaction. Removed immediately after the enzymatic hydrolysis was completed, inactivated, and obtained the enzymatic hydrolysate.

[0008] (2) Take the enzymatic hydrolysate from step (1) into a flask, add ethanol, extract in a boiling water bath, filter while hot to obtain the extract, concentrate by rotary evaporation to obtain the alcohol extract.

[0009] (3) Take the precipitate after filtration in step (2) into a flask, add pure water, extract in a boiling water bath, filter while hot to obtain the extract, and obtain the water extract.

[0010] In one embodiment, the sieving in step (1) is sieving through a 60-100 mesh.

[0011] In one embodiment, the mass ratio of papain, β-glucanase and xylanase in step (1) is 3:2:1.

[0012] In one embodiment, the amount of the complex enzyme added in step (1) is 3-6 wt% of the Polygonatum odoratum powder substrate; preferably 4.8-5%.

[0013] In one embodiment, the mass-to-volume ratio of Polygonatum powder and water in step (1) is 1:15-20, g / mL.

[0014] In one embodiment, the pH value in step (1) is in the range of 5 to 6; preferably 5.5 to 6.

[0015] In one embodiment, the constant temperature conditions in the constant temperature water bath in step (1) are 40-50°C, and the activation time is 5-15 minutes.

[0016] In one embodiment, the enzymatic hydrolysis reaction in step (1) is carried out at a temperature of 40-60°C for 1-2 hours; preferably at a temperature of 50°C for 1.5 hours.

[0017] In one embodiment, the amount of compound enzyme added in step (1) is 5% of the Polygonatum powder, the pH value is 6, the enzymatic hydrolysis temperature is 50°C, and the time is 1.5h.

[0018] In one embodiment, the inactivation in step (1) is performed in a water bath at 100°C for 5 minutes.

[0019] In one embodiment, the ethanol in step (2) is an aqueous solution of ethanol with a volume fraction of 80-90%; preferably, it is an aqueous solution of ethanol with a volume fraction of 80%.

[0020] In one embodiment, the boiling water bath extraction time in step (2) is 1 to 2 hours.

[0021] In one embodiment, the volume-to-mass ratio of ethanol to Polygonatum powder in step (2) is 300-600:1, mL / g.

[0022] Another object of the present invention is to provide an application of the above-described method in the field of deep processing technology of Polygonatum odoratum.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention, through continuous optimization of the types and amounts of compound enzymes, enzymatic hydrolysis time, and enzymatic hydrolysis pH, determined the optimal enzymatic hydrolysis conditions. These conditions resulted in significant changes in the composition of phenolic substances in the enzymatically hydrolyzed Polygonatum odoratum extract, the appearance of various new substances, and good antioxidant capacity, providing a certain scientific basis for the field of deep processing technology of Polygonatum odoratum. Attached Figure Description

[0025] Figure 1 The above are HPLC chromatograms of phenolic substances in the extracts of Polygonatum odoratum obtained in Example 1 and Comparative Examples 1-7 of this invention.

[0026] Figure 2 The HPLC spectra of different phenolic substances in the extract of Polygonatum odoratum are shown.

[0027] Figure 3 The HPLC spectrum of kaempferol-3-O-rutin, a phenolic standard;

[0028] Figure 4 This is a graph showing the total reducing power data of the Polygonatum odoratum extracts obtained in Example 1 and Comparative Examples 1-7 of the present invention. Detailed Implementation

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0030] The determination method involved in this invention:

[0031] 1. Determination of total polysaccharide content

[0032] Preparation of standard curve: Accurately weigh 3.3 mg of anhydrous glucose, add water to make up to 10 mL, and prepare a standard solution of 0.33 mg / mL. Then, pipette 0, 20, 40, 80, 160, and 200 mL of the standard solution into test tubes, add water to make up to 200 μL, add another 200 μL of water, add 1.6 mL of 0.2% anthrone-sulfuric acid solution in an ice-water bath, shake well, seal, boil in a water bath for 10 min, cool to room temperature in an ice-water bath, and measure the absorbance at 582 nm.

[0033] The standard curve for this experiment is Y = 1.8948X - 0.0004, R0 2 =0.9993, where X is the glucose mass concentration, Y is the absorbance, and n = 3.

[0034] Content determination: Accurately pipette 200 mL of standard solution into a test tube, add 200 μL of water, add 1.6 mL of 0.2% anthrone-sulfuric acid solution in an ice-water bath, shake well, seal, boil in a water bath for 10 min, cool to room temperature in an ice-water bath, and measure the absorbance at 582 nm. Each sample needs to be repeated 3 times. Substitute the results into the above standard curve to calculate the polysaccharide content in the sample.

[0035] 2. Determination of total saponin content

[0036] Preparation of standard curve: Accurately weigh 5.0000 mg of sarsaki saponin and dilute to 10 mL with 80% ethanol to prepare a 0.5 g·L⁻¹ standard curve. -1 The standard solution was prepared, and then 50, 100, 200, 300, 400, and 500 μL of the standard solution were accurately pipetted into test tubes. The volume was brought up to 1 mL with 80% ethanol. The solvent was evaporated in a 60°C water bath. 0.5 mL of 5% vanillin-glacial acetic acid solution was added first, followed by 0.8 mL of perchloric acid. The mixture was stirred and incubated in a 60°C water bath for 20 min. The mixture was then cooled in ice water for 30 min. 5 mL of glacial acetic acid was added, and the absorbance was measured at a wavelength of 540 nm.

[0037] The standard curve for this experiment is Y = 1.8652X + 0.0051, R0 2 =0.9998, where X is the mass concentration of sarsaparilla saponin, Y is the absorbance, and n = 3.

[0038] Content determination: Accurately pipette 1 mL of the extract into a test tube, evaporate the solvent in a 60℃ water bath, add 0.5 mL of 5% vanillin-glacial acetic acid solution, then add 0.8 mL of perchloric acid, mix well, incubate in a 60℃ water bath for 20 min, cool in ice water for 30 min, add 5 mL of glacial acetic acid, use 80% methanol as a blank control, and measure the absorbance at a wavelength of 540 nm. Each sample needs to be repeated three times. Substitute the results into the standard curve and calculate the total saponin content in the sample.

[0039] 3. Determination of total phenol content

[0040] Preparation of standard curve: Accurately weigh 10.0000 mg of gallic acid and dilute to 10 mL with 80% ethanol to prepare a standard curve of 1 g·L⁻¹. -1 The standard solution was prepared by accurately pipetting 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution into 10 mL volumetric flasks, respectively. 80% ethanol was added to bring the volume to 2 mL, followed by the accurate addition of 3 mL of 10% Folin-Ciocalteu solution. The solution was then diluted to volume with 7.5% Na₂CO₃ solution, shaken well, and reacted at room temperature in the dark for 30 min. The solution was filtered through a 0.45 μm filter, and the absorbance was measured at 765 nm. The standard curve for this experiment is Y = 10.813X - 0.0008, R₀ = 10.813X - 0.0008. 2=0.9991, where X is the mass concentration of gallic acid, Y is the absorbance, and n = 3.

[0041] Content determination: Take 2 mL of extract, accurately add 3 mL of 10% Folin-Ciocalteu solution, then dilute to volume with 7.5% NaCO3 solution, shake well, react at room temperature in the dark for 30 min, filter through a 0.45 μm filter membrane, and measure the absorbance at 765 nm. Use 80% ethanol as a control and measure the absorbance at 765 nm. Each sample needs to be repeated three times. Substitute the above standard curve to calculate the total phenol content in the sample.

[0042] 4. Determination of phenolic components in Polygonatum odoratum alcohol extract

[0043] The analysis was performed using HPLC, and the specific steps were as follows:

[0044] Preparation of the test solution: The alcohol extract was lyophilized. 10 mg of the sample alcohol extract was weighed into a 2 mL centrifuge tube, dissolved in 50% methanol to prepare a 20 mg / mL sample solution. The solution was shaken well and filtered through a 0.22 μm filter membrane. Chromatographic conditions: Liquid chromatograph: Aglient 1260 high-performance liquid chromatography system; Column: XDB C18 (5 μm, 4.6 mm × 250 mm); Column temperature: 40℃; Injector temperature: 25℃; Injection volume: 10 μL; Flow rate: 1 mL / min; Detector: DAD; Mobile phase: A - 0.1% formic acid aqueous solution, B - 95% methanol (containing 0.1% formic acid); Elution program is shown in Table 1.

[0045] Table 1. Gradient elution programs for high performance liquid chromatography

[0046]

[0047] 5. Determination of antioxidant activity of Polygonatum odoratum alcohol extract

[0048] Preparation of standard curve: 0.3 mol·L -1 Acetate buffer, 10 mmol / L -1 TPTZ solution and 20 mmol·L -1 Ferric chloride solution mixed in a 10:1:1 ratio constitutes the FRAP working solution. (1.6 mmol / L) -1 Ferrous sulfate was used as the standard solution. 10, 20, 30, 40, 50, 60, 80, and 100 μL of the standard solution were respectively pipetted into test tubes, and water was added to make up to 100 μL. 900 μL of FRAP working solution was added, mixed well, and reacted at 37°C in the dark for 10 min. The absorbance was measured at 593 nm.

[0049] The standard curve for this experiment is Y = 0.9803X - 0.0028, R² = 0.9997, where X is the molar concentration of ferrous sulfate, Y is the absorbance, and n = 3.

[0050] Activity assay: Accurately pipette 100 μL of sample solution into a test tube, mix with 900 μL of FRAP working solution, and react at 37 °C in the dark for 10 min. Measure the absorbance at 593 nm. Each sample should be tested three times and the results should be included in the standard curve above. The total reducing power of the Polygonatum odoratum extract is expressed as the micromoles of ferrous sulfate equivalent per gram of dry weight of Polygonatum odoratum powder (μmolFE / g DW).

[0051] The source and activity of the enzymes involved in this invention:

[0052] Cellulase (50 U / mg, biotechnology grade, CAS: 9012-54-8), papain (>200 U / mg, CAS: 9001-73-4), pectinase (derived from Aspergillus niger, 30,000 U / g, CAS: 9032-75-1), β-glucanase (30,000 U / g, 98%, CAS: 9025-70-1), and xylanase (100,000 U / g, CAS: 9025-57-4) were all purchased from Maclean's.

[0053] Example 1

[0054] An efficient enzymatic hydrolysis method for releasing phenolic substances specifically includes the following steps:

[0055] (1) Clean the Polygonatum sibiricum, slice and dry it, pass it through a 60-mesh sieve, accurately weigh 0.25g of Polygonatum sibiricum powder and 5% of the substrate mass of the complex enzyme (papain:β-glucanase:xylanase mass ratio = 3:2:1), add it to 5mL of water, and adjust the pH value to 6 with 0.1mol / L HCl. Activate it in a 40℃ constant temperature water bath for 10min, and then enzymatically hydrolyze it at 50℃ for 1.5h. After the enzymatic hydrolysis is completed, take it out immediately and place it in a 100℃ water bath for 5min to inactivate it.

[0056] (2) Take the enzymatic hydrolysate from step (1) into a 250mL round-bottom flask, add 150mL of 80% ethanol, boil in a water bath for 1h, filter while hot, and wash the round-bottom flask three times with 10mL of 80% hot ethanol. Combine the filtrates, concentrate by rotary evaporation, and make up to 25mL to obtain the alcohol extract.

[0057] (3) Place the precipitate filtered in step (2) into a 250mL round-bottom flask, add 150mL of water, boil in a water bath for 1 hour, filter while hot, and wash the round-bottom flask three times with 10mL of hot water. Combine the filtrates and make up to 250mL to obtain the water extract.

[0058] Example 2

[0059] The only difference from Example 1 is that step (1) is carried out according to the conditions in Table 2 below, while other parameters and conditions are the same as in Example 1.

[0060] Table 2. Enzymatic hydrolysis conditions

[0061] Enzyme addition amount (%) Enzymatic hydrolysis time (h) Enzymatic pH 3.6 1.5 5 3.6 1.5 6 3.6 1 5.5 3.6 2 5.5 4.8 2 6 4.8 1 6 5 (Example 1) 1.5 6 4.8 2 5 4.8 1 5 6 2 5.5 6 1 5.5 6 1.5 6 6 1.5 5

[0062] Comparative Example 1

[0063] The only difference from Example 1 is that the complex enzyme in step (1) is replaced with a pectinase:β-glucanase:xylanase mass ratio of 3:2:1, while the other parameters and conditions are the same as in Example 1.

[0064] Comparative Example 2

[0065] The only difference from Example 1 is that the complex enzyme in step (1) is replaced with a papain:β-glucanase:cellulase mass ratio of 3:2:1, and all other parameters and conditions are the same as in Example 1.

[0066] Comparative Example 3

[0067] The only difference from Example 1 is that in step (1), the complex enzyme is replaced with a mass ratio of β-glucanase to xylanase of 2:1. All other parameters and conditions are the same as in Example 1.

[0068] Comparative Example 4

[0069] The only difference from Example 1 is that the complex enzyme in step (1) is replaced with papain:β-glucanase in a mass ratio of 3:2. All other parameters and conditions are the same as in Example 1.

[0070] Comparative Example 5

[0071] The only difference from Example 1 is that the complex enzyme in step (1) is replaced with papain:β-glucanase:xylanase:pectinase in a mass ratio of 3:2:1:1. All other parameters and conditions are the same as in Example 1.

[0072] Comparative Example 6

[0073] The only difference from Example 1 is that the complex enzyme in step (1) is replaced with a mass ratio of papain:β-glucanase:xylanase:cellulase = 3:2:1:1, and all other parameters and conditions are the same as in Example 1.

[0074] Comparative Example 7

[0075] The only difference from Example 1 is that the addition of the compound enzyme is omitted, while all other parameters and conditions are the same as in Example 1.

[0076] Results Measurement

[0077] 1. The components of the alcoholic and aqueous extracts obtained in the examples and comparative examples were determined, and the results are shown in Tables 3 and 4:

[0078] Table 3. Content of extracts in the examples

[0079]

[0080]

[0081] The results of treatment with different enzyme dosages, hydrolysis times, and hydrolysis pH are shown in Table 3. Compared with the control group (Comparative Example 7), which contained 155.29±6.16 mg / g of total polysaccharides, 106.50±5.17 mg / g of total saponins, and 1.80±0.09 mg / g of total phenols, the polysaccharides in Polygonatum were degraded to varying degrees after compound enzyme treatment. This indicates that Polygonatum polysaccharides have poor stability and are easily degraded by enzymes, suggesting that Polygonatum polysaccharides can be effectively degraded by enzymes in vivo to obtain nutrients. The contents of total saponins and total phenols increased by 7.98%–46.48% and 30.69%–51.82%, respectively, indicating that compound enzyme treatment has a significant effect on the release of small molecules in Polygonatum. The increase in total phenol content may be due to the action of compound enzymes, which release some phenolic substances from large molecules such as proteins and cellulose. Considering the content of the three components, the total polysaccharide obtained from the treatment in Example 1 had a lower degree of degradation, the highest total saponin content, and a relatively high total phenol content, resulting in the best overall effect.

[0082] Table 4. Content of extracts in the examples and comparative examples

[0083]

[0084] As shown in Table 4, the contents of total polysaccharides, total saponins, and total phenols measured in Example 1 were all higher than those in Comparative Examples 1, 2, and 6. Compared with Comparative Examples 3 and 4, the polysaccharide content in Example 1 was slightly lower, but the total saponin content was significantly increased. Compared with Comparative Example 4, the polysaccharide degradation degree in Example 1 was greater, but the release of small molecules was also greater, resulting in a significant increase in the contents of total saponins and total phenols. This indicates that the compound enzyme treatment of Polygonatum sibiricum in Example 1 has a certain synergistic effect and can effectively promote the release of phenolic substances in Polygonatum sibiricum.

[0085] 2. The alcohol extracts obtained in Example 1 and Comparative Examples 1-7 were analyzed by HPLC to determine the changes in phenolic components. The results are as follows: Figure 1 As shown in Table 5:

[0086] Table 5. Peak areas of polysaccharide substances in HPLC (mAU·min)

[0087] Peak number Comparative Example 7 Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 1 31.23 31.42 15.38 25.79 34.02 32.92 19.87 21.68 2 50.07 66.40 62.47 57.15 74.88 68.15 68.14 61.60 3 21.72 28.40 21.50 25.10 24.71 18.78 20.27 23.19 4 58.18 58.48 53.96 57.73 57.24 60.46 56.23 50.38 5 6.72 11.57 7.04 10.26 12.40 11.84 11.37 10.52 6 11.26 13.22 11.73 11.61 12.63 11.54 14.76 15.35 7 33.41 45.00 36.38 47.23 41.19 52.02 50.41 47.59 8 9.19 6.32 7.88 5.26 5.69 8.08 5.17 5.46 9 6.67 7.34 12.87 11.76 12.84 7.05 10.85 9.21 10 5.15 8.13 9.18 10.97 11.56 6.95 9.41 9.39 11 12.12 12.39 12.79 10.13 12.05 8.92 12.81 12.17 12 11.08 11.51 11.90 11.35 10.91 10.59 14.94 12.83 13* - 10.59 - 15.97 - 12.44 14.96 11.45 14* - 8.37 - 5.69 - 5.44 7.89 7.13 15* - 10.61 - 11.76 - 10.98 12.62 9.96 16* - 18.97 14.77 10.32 28.33 10.70 20.52 15.67

[0088] Note: All the above results were measured at a wavelength of 280nm. "*" indicates newly appearing phenolic substances, and "-" indicates that no corresponding peaks appeared.

[0089] Phenolic substances, due to their benzene ring π-electron conjugated system, can absorb shorter wavelengths of ultraviolet light. However, under the influence of hydroxyl groups and other substituents, their maximum absorption wavelength will vary. Most phenolic substances exhibit ultraviolet absorption at a wavelength of 280 nm. HPLC analysis revealed that, for example... Figure 1 As shown, Comparative Example 7 contains at least 12 phenolic substances, indicating that the phenolic composition of Polygonatum is relatively complex. The spectrum corresponding to the peak positions is as follows. Figure 2 As shown, ultraviolet absorption is observed around 280 nm, which is consistent with the basic absorption characteristics of phenolic substances. Taking substance 12 as an example, through comparison with standards and sample addition analysis, it was found that the retention times of substance 12 and kaempferol-3-O-rutin were 35.213 min and 35.121 min, respectively, and ultraviolet absorption was observed near 250 nm, 300 nm, and 350 nm. After adding standards in Example 1, the peak area of ​​this substance increased, and substance 12 can be identified as kaempferol-3-O-rutin.

[0090] After treatment with the compound enzyme, the phenolic substances in Polygonatum showed varying degrees of increased diversity and peak area. Firstly, the most obvious change was the appearance of new peaks, indicating the detection of new phenolic substances. As shown in Table 5, compared to Comparative Examples 1, 3, and 7, three to four new phenolic substances were detected in Example 1, indicating the presence of bound polyphenols in Polygonatum and the effective release of these by the compound enzyme. Secondly, the content of phenolic substances increased. Combining the peak area changes in Table 5 and the total phenolic content results in Table 4, a comparison of Example 1 with Comparative Examples 2, 4, 5, and 6 revealed that after treatment with the compound enzyme in Example 1, most phenolic components showed a significant increase, indicating that the compound enzyme in Example 1 effectively promoted the release of phenolic substances from Polygonatum. All these results demonstrate that the compound enzyme of this application can effectively promote the release of bound phenolic substances from Polygonatum.

[0091] 3. The antioxidant activity of the alcohol extracts obtained in Example 1 and Comparative Examples 1-7 was determined, and the results are shown in Table 6. Figure 4 As shown:

[0092] Table 6. Antioxidant activity of alcohol extracts obtained in examples and comparative examples

[0093]

[0094] Phenolic substances possess good antioxidant activity. (Combined) Figure 4Table 4 shows that, except for Comparative Example 1, the total reducing power of the Polygonatum odoratum extract treated with other compound enzymes was higher than that of the control group (Comparative Example 7), with Example 1 showing the highest and best effect. Moreover, it was found that the total reducing power was positively correlated with the total phenol content, indicating that the efficient release of phenolic substances can increase its antioxidant activity.

[0095] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A highly efficient enzymatic hydrolysis method for releasing polysaccharide substances from Polygonatum, characterized in that, The method includes the following steps: (1) Powdered Polygonatum sibiricum was sieved, and the powdered Polygonatum sibiricum and the complex enzyme composed of papain, β-glucanase and xylanase were weighed and added to water. The pH value was adjusted, and the enzyme was first activated in a constant temperature water bath. Then the enzymatic hydrolysis reaction was carried out. After the enzymatic hydrolysis was completed, the enzyme was immediately removed and inactivated to obtain the enzymatic hydrolysate. The mass ratio of papain, β-glucanase and xylanase was 3:2:

1. The pH value range was 5 to 6. (2) Take the enzymatic hydrolysate from step (1) into a flask, add ethanol, extract in a boiling water bath, filter while hot to obtain the extract, concentrate by rotary evaporation to obtain the alcohol extract. (3) Take the precipitate after filtration in step (2) into a flask, add pure water, extract in a boiling water bath, filter while hot to obtain the extract, and obtain the water extract.

2. The method according to claim 1, characterized in that, The amount of the complex enzyme added in step (1) is 3-6 wt% of the Polygonatum odoratum powder substrate.

3. The method according to claim 1, characterized in that, The mass-to-volume ratio of Polygonatum powder and water in step (1) is 1:15-20, g / mL.

4. The method according to claim 1, characterized in that, In step (1), the constant temperature conditions in the constant temperature water bath are 40-50℃, and the activation time is 5-15 minutes.

5. The method according to claim 1, characterized in that, The enzymatic hydrolysis reaction in step (1) is carried out at a temperature of 40–60°C for 1–2 hours.

6. The method according to claim 1, characterized in that, The ethanol mentioned in step (2) is an aqueous solution of ethanol with a volume fraction of 80-90%.

7. The method according to claim 1, characterized in that, The volume-to-mass ratio of ethanol to Polygonatum powder in step (2) is 300-600:1, mL / g.

8. The application of the method described in any one of claims 1 to 7 in the field of deep processing technology of Polygonatum odoratum.

Images