A leafy green herb bioactive polypeptide, its preparation method and functional products

Abstract

This invention relates to the technical field of deep processing of leafy greens, and particularly to an active polypeptide from leafy greens, its preparation method, and functional products. The preparation method involves sequentially hydrolyzing leafy greens with β-glucanase, xylanase, pepsin, and trypsin. The resulting solution is then centrifuged, filtered, ultrafiltered, and nanofiltered to obtain a leafy green polypeptide solution with a molecular weight between 200 and 3000 Da. The preparation method provided by this invention not only significantly improves the yield of the active polypeptide from leafy greens, but also yields active polypeptides with significant pancreatic lipase inhibitory activity, anti-glycation activity (anti-AGEs formation), sleep-aiding function, and beneficial bacteria-promoting activity.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology of leafy plants, and in particular to an active polypeptide of leafy plants, its preparation method, and functional products. Background Technology

[0002] Leafy grass, also known as protein grass, contains eighteen kinds of amino acids, vitamin E, ascorbic acid, β-carotene, chlorophyll, isoflavones, SOD, and trace elements and minerals such as calcium, iron, zinc, and selenium, and is also rich in protein. Currently, most research on leafy grass focuses on its role in soil improvement and as a protein feed, with few reports on the deep processing of leafy grass to prepare bioactive peptides. Patent document CN115368434A (publication date November 22, 2022) discloses a high-SOD-content leafy grass bioactive peptide and its preparation method. This method utilizes acidic and neutral proteases to enzymatically hydrolyze leafy grass, followed by secondary separation, membrane treatment, enzyme inactivation, and drying to obtain leafy grass peptides with high SOD content; however, its related activities have not been studied. Summary of the Invention

[0003] To address the problems mentioned in the background section, this invention provides a method for preparing bioactive polypeptides from leafy greens, the technical solution of which is as follows:

[0004] The preparation method of this herbaceous plant bioactive polypeptide includes the following steps:

[0005] Raw material pretreatment: Crush the leafy grass into powder, then add water and stir to obtain a slurry;

[0006] First hydrolysis: β-glucanase is added to the slurry for the first hydrolysis. After the hydrolysis is completed, the enzyme is inactivated to obtain the first hydrolysate.

[0007] Second hydrolysis: Xylanase is added to the first hydrolysate for a second hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the second hydrolysate.

[0008] Third hydrolysis: Pepsin is added to the second hydrolysate for a third hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the third hydrolysate.

[0009] Fourth hydrolysis: Trypsin is added to the third hydrolysate for a fourth hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the fourth hydrolysate.

[0010] The fourth hydrolysate is subjected to solid-liquid separation to obtain a supernatant containing the active peptide of *Gnaphalium affine*. The supernatant is then filtered to retain the active peptide of *Gnaphalium affine*, thus obtaining the active peptide of *Gnaphalium affine*.

[0011] This invention is the first to utilize β-glucanase, xylanase, pepsin, and trypsin to sequentially enzymatically hydrolyze edible grass, thereby preparing edible grass polypeptides with significant pancreatic lipase inhibitory activity, anti-glycation activity (anti-AGEs formation), sleep-aiding function, and beneficial bacteria-promoting activity.

[0012] In the raw material pretreatment step, the mass ratio of the leafy grass to water is 1:(10-20).

[0013] In the first hydrolysis step, β-glucanase is added to the slurry, the pH of the system is adjusted to 4.4-5.1, and hydrolysis is carried out at 45-53℃ for 45-80 min; then the enzyme is inactivated at 85-90℃ for 10-20 min to obtain the first hydrolysate.

[0014] In the second hydrolysis step, xylanase is added to the first hydrolysate to adjust the pH of the system to 3.7-4.2, and hydrolysis is carried out at 45-53°C for 45-80 min; then the enzyme is inactivated at 85-90°C for 10-20 min to obtain the second hydrolysate.

[0015] In the third hydrolysis step, pepsin is added to the second hydrolysate to adjust the pH of the system to 2.7-3.2, and hydrolysis is carried out at 34-38°C for 100-150 min; then enzyme inactivation treatment is carried out at 85-90°C for 10-20 min to obtain the third hydrolysate.

[0016] In the fourth hydrolysis step, trypsin is added to the third hydrolysate to adjust the pH of the system to 7.5-8.3, and hydrolysis is carried out at 34-38℃ for 50-70 min; then enzyme inactivation treatment is carried out at 85-90℃ for 10-20 min to obtain the fourth hydrolysate.

[0017] Based on the protein content in the edible grass, the amount of β-glucanase added is 200-400 U / g; the amount of xylanase added is 150-300 U / g; the amount of pepsin added is 2000-4000 U / g; and the amount of trypsin added is 4000-7000 U / g.

[0018] The fourth hydrolysate was centrifuged at 4000–6000 rpm for 8–15 min to obtain a supernatant; the pH of the supernatant was adjusted to 5.7–6.1, and then subjected to microfiltration through a membrane with a pore size of 0.22–0.45 μm and ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da; the ultrafiltration peptide solution was concentrated using a 200 Da nanofiltration membrane device and then sterilized to obtain a peptide solution with a molecular weight of 200–3000 Da, which is the final product.

[0019] In one embodiment, the polypeptide liquid with a molecular weight of 200-3000 Da is concentrated to a polypeptide mass content of 6%-10%, and the concentrate is made into powder.

[0020] The present invention also provides a leafy herb active polypeptide, which is prepared by the above-mentioned method for preparing leafy herb active polypeptide.

[0021] The present invention also provides a functional product, the components of which include the above-mentioned active polypeptides of edible leaves;

[0022] The functional product includes at least one of the following functions:

[0023] (1) It possesses pancreatic lipase inhibitory activity;

[0024] (2) It possesses sleep-aiding activity;

[0025] (3) It possesses anti-glycation activity;

[0026] (4) It possesses antioxidant properties;

[0027] (5) It has beneficial bacteria promoting activity.

[0028] In one embodiment, the functional product includes a pharmaceutical product.

[0029] Based on the above, compared with the prior art, the method for preparing bioactive polypeptides from leafy greens provided by the present invention has the following beneficial effects:

[0030] This invention first uses β-glucanase and xylanase to pre-treat the leafy grass with enzymatic hydrolysis, which may improve the fluidity of the subsequent hydrolysate and the enzymatic hydrolysis yield by promoting the release of intracellular proteins and reducing the viscosity of the hydrolysate, respectively. Then, pepsin and trypsin are used to further hydrolyze the hydrolysate to obtain the active peptides of leafy grass.

[0031] The active polypeptide of edible leaves prepared by this invention has lipid-lowering activity, anti-glycation activity (anti-AGEs formation), sleep aid and probiotic functions, and can be used as a raw material for regulating intestinal health, lowering lipids, aiding sleep and preventing skin aging. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0034] This invention provides an operational example of a method for preparing bioactive polypeptides from leafy greens, the specific steps of which are as follows:

[0035] Step 1: Raw material pretreatment:

[0036] The leafy grass is crushed into powder, then water is added and stirred to obtain a slurry; wherein the mass ratio of the leafy grass powder to water is 1:(10-20).

[0037] Step 2, First Hydrolysis:

[0038] β-glucanase is added to the slurry for the first hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the first hydrolysate; specifically:

[0039] β-glucanase was added to the slurry to adjust the pH of the system to 4.4–5.1, and hydrolyzed at 45–53°C for 45–80 min; then, the enzyme was inactivated at 85–90°C for 10–20 min to obtain the first hydrolysate. The amount of β-glucanase added was 200–400 U / g, based on the protein content of the edible grass.

[0040] Step 3, Second Hydrolysis:

[0041] Xylanase was added to the first hydrolysate for a second hydrolysis. After hydrolysis, the enzyme was inactivated to obtain the second hydrolysate. Specifically:

[0042] Xylanase was added to the first hydrolysate to adjust the pH of the system to 3.7–4.2, and hydrolyzed at 45–53°C for 45–80 min; then, the enzyme was inactivated at 85–90°C for 10–20 min to obtain the second hydrolysate. The amount of xylanase added was 150–300 U / g, based on the protein content of the edible grass.

[0043] Step 4, Third Hydrolysis:

[0044] Pepsin was added to the second hydrolysate for a third hydrolysis. After hydrolysis, the enzyme was inactivated to obtain the third hydrolysate. Specifically:

[0045] Pepsin was added to the second hydrolysate to adjust the pH of the system to 2.7–3.2, and hydrolysis was carried out at 34–38°C for 100–150 min; then, the enzyme was inactivated at 85–90°C for 10–20 min to obtain the third hydrolysate. The amount of pepsin added was 2000–4000 U / g, based on the protein content of the herb.

[0046] Step 5, Fourth Hydrolysis:

[0047] Trypsin is added to the third hydrolysate for a fourth hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the fourth hydrolysate; specifically:

[0048] Trypsin was added to the third hydrolysate to adjust the pH of the system to 7.5–8.3, and hydrolysis was carried out at 34–38°C for 50–70 min; then, the enzyme was inactivated at 85–90°C for 10–20 min to obtain the fourth hydrolysate. The amount of trypsin added was 4000–7000 U / g, based on the protein content of the leafwort.

[0049] Step Six: Separation and Filtration

[0050] The fourth hydrolysate was centrifuged to obtain a supernatant containing active peptides from edible leaves; the pH of the supernatant was adjusted to 5.7-6.1, and then subjected to microfiltration through a 0.22-0.45 micrometer membrane and ultrafiltration through a 3000 Da membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da.

[0051] The centrifugation treatment conditions for the fermentation broth are centrifugation at 4000-6000 rpm for 8-15 min.

[0052] Step 7: Nanofiltration concentration and sterilization:

[0053] The ultrafiltration peptide solution was concentrated using a 200 Da nanofiltration membrane device and then sterilized to obtain a peptide solution with a molecular weight of 200–3000 Da.

[0054] Step 8: Powdering

[0055] The polypeptide solution with a molecular weight of 200-3000 Da is concentrated to a polypeptide content of 6%-10%, and the concentrate is then made into powder. The concentrate may be spray-dried to form powder.

[0056] The present invention provides the following embodiments and comparative examples.

[0057] Example 1

[0058] S100. Raw material pretreatment: Crush the leafy grass into powder (protein content: 22%), then take 300g of leafy grass powder, add 4.5kg of water to soften it, stir for 30min until completely mixed to form leafy grass slurry.

[0059] S200, First hydrolysis: Add 20KU of β-glucanase to the slurry, adjust the pH of the slurry to 4.8, hydrolyze at 50℃ for 60min; inactivate the enzyme at 90℃ for 10min to obtain the first hydrolysate.

[0060] S300, Second Hydrolysis: Add 13 KU of xylanase to the first hydrolysate, control the pH to 4.0, and hydrolyze at 50℃ for 60 min. After the second hydrolysis, heat to 85℃ to inactivate the enzyme for 20 min to obtain the second hydrolysate.

[0061] S400, Third hydrolysis: Add 198 KU of pepsin to the second hydrolysate, control the pH to 3.0, and hydrolyze at 37℃ for 120 min. After hydrolysis, raise the temperature to 85℃ for 20 min to inactivate the enzyme and obtain the third hydrolysate.

[0062] S500, Fourth Hydrolysis: Add 330 KU of trypsin to the third hydrolysate, control the pH to 8.0, and hydrolyze at 37℃ for 60 min. After hydrolysis, heat to 90℃ for 10 min to inactivate the enzyme and obtain the fourth hydrolysate.

[0063] S600, centrifugation, microfiltration and ultrafiltration: The fourth hydrolysate was centrifuged at 4000 rpm for 15 min to obtain the supernatant. After adjusting the pH to 5.9, the supernatant was subjected to microfiltration through a 0.45-micron membrane and ultrafiltration through a 3000 Da membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da.

[0064] S700, Nanofiltration Concentration and Sterilization: The ultrafiltration peptide solution is concentrated to a peptide content of 10% using a 200Da nanofiltration membrane device, and then sterilized to obtain a concentrated peptide solution with a molecular weight of 200-3000Da.

[0065] Example 2

[0066] S100. Raw material pretreatment: Crush the leafy grass into powder (protein content: 22%), then take 300g of leafy grass powder, add 3kg of water to soften it, stir for 30min until completely mixed to form leafy grass slurry.

[0067] S200, First hydrolysis: Add 26KU of β-glucanase to the slurry, adjust the pH of the slurry to 5.1, hydrolyze at 53℃ for 45min; inactivate the enzyme at 85℃ for 20min to obtain the first hydrolysate.

[0068] S300, Second Hydrolysis: Add 19 KU of xylanase to the first hydrolysate, control the pH to 4.2, and hydrolyze at 45℃ for 80 min. After the second hydrolysis, heat to 85℃ to inactivate the enzyme for 20 min to obtain the second hydrolysate.

[0069] S400, Third hydrolysis: Add 264 KU of pepsin to the second hydrolysate, control the pH to 3.2, and hydrolyze at 34℃ for 100 min. After hydrolysis, heat to 90℃ for 10 min to inactivate the enzyme and obtain the third hydrolysate.

[0070] S500, Fourth Hydrolysis: Add 264 KU of trypsin to the third hydrolysate, control the pH to 7.5, and hydrolyze at 38℃ for 50 min. After hydrolysis, heat to 90℃ for 10 min to inactivate the enzyme and obtain the fourth hydrolysate.

[0071] S600, centrifugation, microfiltration and ultrafiltration: The fourth hydrolysate was centrifuged at 6000 rpm for 8 min to obtain the supernatant. After adjusting the pH to 6.1, the supernatant was subjected to microfiltration through a 0.45-micron membrane and ultrafiltration through a 3000 Da membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da.

[0072] S700, Nanofiltration Concentration and Sterilization: The ultrafiltration peptide solution is concentrated to a peptide content of 6% using a 200Da nanofiltration membrane device, and then sterilized to obtain a concentrated peptide solution with a molecular weight of 200-3000Da.

[0073] Example 3

[0074] S100. Raw material pretreatment: Crush the leafy grass into powder (protein content: 22%), then take 300g of leafy grass powder, add 6kg of water to soften it, stir for 30min until completely mixed to form leafy grass slurry.

[0075] S200, First hydrolysis: Add 13.2 KU of β-glucanase to the slurry, adjust the pH of the slurry to 4.4, and hydrolyze at 45℃ for 80 min; inactivate the enzyme at 85℃ for 20 min to obtain the first hydrolysate.

[0076] S300, Second Hydrolysis: Add 10 KU of xylanase to the first hydrolysate, control the pH to 3.7, and hydrolyze at 53℃ for 45 min. After the second hydrolysis, heat to 90℃ to inactivate the enzyme for 10 min to obtain the second hydrolysate.

[0077] S400, Third hydrolysis: Add 132 KU of pepsin to the second hydrolysate, control the pH to 2.7, and hydrolyze at 38℃ for 150 min. After hydrolysis, heat to 90℃ for 10 min to inactivate the enzyme and obtain the third hydrolysate.

[0078] S500, Fourth Hydrolysis: Add 462 KU of trypsin to the third hydrolysate, control the pH to 8.3, and hydrolyze at 34℃ for 70 min. After hydrolysis, heat to 85℃ for 20 min to inactivate the enzyme and obtain the fourth hydrolysate.

[0079] S600, centrifugation, microfiltration and ultrafiltration: The fourth hydrolysate was centrifuged at 5000 rpm for 10 min to obtain the supernatant. After adjusting the pH to 5.7, the supernatant was subjected to microfiltration through a 0.45-micron membrane and ultrafiltration through a 3000 Da membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da.

[0080] S700, Nanofiltration Concentration and Sterilization: The ultrafiltration peptide solution is concentrated to a peptide content of 8% using a 200Da nanofiltration membrane device, and then sterilized to obtain a concentrated peptide solution with a molecular weight of 200-3000Da.

[0081] Comparative Example 1

[0082] The β-glucanase in step S200 of Example 1 was replaced with xylanase of equal activity, and all other operations and processes were the same as in Example 1.

[0083] Comparative Example 2

[0084] The xylanase in step S300 of Example 1 was replaced with β-glucanase of equal enzyme activity, and all other operations and processes were the same as in Example 1.

[0085] Comparative Example 3

[0086] In Example 1, the β-glucan and xylanase in steps S200 and S300 were replaced with pepsin of equal enzyme activity, and all other operations and processes were the same as in Example 1.

[0087] Comparative Example 4

[0088] In Example 1, the trypsin in step S500 was replaced with papain of equal activity, and the enzymatic hydrolysis conditions were adjusted to 55°C and pH 6.0 for 60 min. All other operations and processes were the same as in Example 1.

[0089] Comparative Example 5

[0090] In Example 1, the trypsin in step S500 was replaced with bromelain of equal activity, and the enzymatic hydrolysis conditions were adjusted to 60°C and pH 7.5 for 60 min. All other operations and processes were the same as in Example 1.

[0091] In the examples and comparative examples, β-glucanase, xylanase, pepsin, and trypsin were all commercially available enzyme preparations.

[0092] The performance of the leafy green bioactive peptides prepared in the above examples and comparative examples was tested:

[0093] 1. Determination of pancreatic lipase inhibitory activity of bioactive peptides from *Hedyotis diffusa*

[0094] The polypeptide solutions obtained in Examples 1 to 3 and Comparative Examples 1 to 5 were diluted to 10 mg / ml (using water as the solvent), and the pancreatic lipase inhibitory activity was determined using the GB / T 23535-2009 method for lipase activity. The specific results are shown in Table 1.

[0095] Table 1. Results of pancreatic lipase inhibitory activity assays in the examples and comparative cases.

[0096]

[0097] As shown in Table 1, compared with Comparative Example 3, the addition of xylanase or β-glucanase to Comparative Examples 1-2 slightly increased the pancreatic lipase inhibition rate of the active peptides from *Eclipta prostrata*, but the results were significantly lower than those in Examples 1-3. This indicates that the combined enzymatic hydrolysis of β-glucanase and xylanase may significantly improve hydrolysis efficiency by promoting the release of raw protein and reducing the viscosity of the hydrolysate, thereby producing more active peptides from *Eclipta prostrata* with high pancreatic lipase inhibitory activity.

[0098] Compared with Comparative Examples 4 and 5, when trypsin was replaced with papain and bromelain respectively, the trypsin inhibitory activity was significantly lower than that of the Example. This result indicates that the type of enzyme has a significant impact on the activity of phytoesophageal peptides, and that phytoesophageal active peptides with high pancreatic lipase inhibitory activity can be obtained by sequentially enzymatically digesting with β-glucanase, xylanase, pepsin, and trypsin.

[0099] 2. Determination of the growth activity of bioactive peptides from *Lactobacillus plantarum* (CGMCC NO.16436)

[0100] The polypeptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (using water as the solvent), and the growth-promoting effect of yam polypeptides on probiotic growth was measured using the method described in "Study on the In Vitro Growth-Promoting Effect of Probiotics on yam Polypeptides". The specific operation and calculation formula are as follows:

[0101] First, a 10 mL LMR (Liquid Monoclonal Antibiotic Recombinant Human) liquid medium containing 300 μL (approximately 900 million viable bacteria) of *Lactobacillus plantarum* was prepared. Then, 500 μL of a 10 mg / mL solution of *Lactobacillus plantarum* active peptides was added to the experimental group, while 500 μL of sterile water was added to the control group. The cultures were incubated at 37°C for 24 h. After incubation, the culture medium was diluted 20-fold and the OD (Organic Demand) was measured. 600 Calculate the probiotic promotion rate.

[0102] Probiotic promotion rate % = (BA) / A x 100%

[0103] Note: A: OD of the blank group 600 B: OD of the experimental group 600 .

[0104] Lactobacillus plantarum BXM2 was deposited at the China General Microbiological Culture Collection Center on September 6, 2018, with the accession number CGMCC NO.16436; technicians can obtain it from the collection center based on the accession number.

[0105] The formula for MRS liquid culture medium is as follows: 10.0 g beef extract, 20.0 g glucose, 10.0 g tryptone, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate tetrahydrate, 1.0 L deionized water, pH 6.5.

[0106] The specific results obtained by the above experimental methods are as follows:

[0107] Table 2. Results of growth activity assay of bioactive peptides from *Lactobacillus plantarum* (CGMCC NO.16436).

[0108]

[0109] Note: - indicates not detected, which means that it has no effect on promoting the growth of beneficial bacteria. In other words, compared with the control group, it does not promote the growth of beneficial bacteria, that is, its growth rate is less than or equal to 0.

[0110] As shown in Table 2, compared with the examples, no activity promoting the proliferation of beneficial bacteria was found in Comparative Examples 1 to 3, indicating that the synergistic effect of β-glucanase and xylanase can prepare leafy grass polypeptides with the activity of promoting the growth of Lactobacillus plantarum (CGMCC NO.16436).

[0111] Compared to the examples, in Comparative Examples 4 and 5, where trypsin was replaced with equal amounts of papain and bromelain, only Comparative Example 4 showed slight probiotic activity, but it was significantly lower than that of the examples. These results indicate that the combined enzymatic hydrolysis of β-glucanase, xylanase, pepsin, and trypsin is the optimal combination for obtaining leafy herb polypeptides with activity promoting the growth of *Lactobacillus plantarum* (CGMCC NO. 16436).

[0112] 3. Determination of the antioxidant activity of bioactive peptides from leafy green plants

[0113] The peptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (using water as the solvent), and the DPPH and ABTS scavenging rates were determined according to GB / T39100-2020 peptide antioxidant activity assay. The hydroxyl radical scavenging rate was determined using the salicylic acid method. The specific results are shown in Table 3.

[0114] Table 3. Results of antioxidant activity assays for the examples and comparative examples.

[0115]

[0116] Note: -- indicates no inhibitory activity.

[0117] As shown in Table 3, the ABST scavenging activity and hydroxyl radical scavenging activity of Examples 1-3 were significantly higher than those of Comparative Examples 1-3, and there was no DPPH scavenging activity in Comparative Example 3. This indicates that the combined enzymatic hydrolysis of β-glucanase and xylanase can obtain edible leafy herb active peptides with high antioxidant activity.

[0118] Comparing Comparative Examples 4-5 with Example 1, the results showed that the DPPH scavenging activity of Comparative Examples 4 and 5 decreased by 38.02% and 40.51%, respectively. In contrast, the herbaceous plant active peptides obtained by trypsin hydrolysis in Example 1 exhibited significant activity in scavenging DPPH, ABTS, and hydroxyl radicals. These results indicate that a combination of β-glucanase, xylanase, pepsin, and trypsin can yield herbaceous plant active peptides with high antioxidant activity.

[0119] 4. Assay of the sleep-aiding activity of bioactive peptides from *Hedyotis diffusa*

[0120] The polypeptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (using water as the solvent), and the γ-aminobutyric acid (GABA) content was determined using the method for the determination of γ-aminobutyric acid (GABA) in QB / T5633.7-2022. The specific results are shown in Table 4.

[0121] Table 4. Results of γ-aminobutyric acid content determination in the examples and comparative examples.

[0122]

[0123] As shown in Table 4, compared with Example 1, there was no significant difference in the γ-aminobutyric acid (GABA) levels in Examples 2 and 3, while the GABA concentrations in Comparative Examples 1-3 were significantly decreased, with Comparative Example 3 having the lowest GABA concentration. This indicates that the combined enzymatic hydrolysis of β-glucanase and xylanase may increase the GABA level of the active polypeptides of *Gynostemma pentaphyllum* by promoting the release of cell contents.

[0124] Furthermore, compared to Example 1, the concentration of γ-aminobutyric acid (GABA) in Examples 4 and 5 was significantly reduced after replacing trypsin with papain and bromelain. This result indicates that a complex enzymatic hydrolysis using β-glucanase, xylanase, pepsin, and trypsin can yield bioactive peptides from leafy greens with high GABA levels.

[0125] 5. Determination of the anti-glycation activity of bioactive peptides from *Hedyotis diffusa*

[0126] The polypeptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (using water as the solvent), and the anti-AGEs formation assay method described in the article "Response Surface Optimization Enzymatic-Assisted Extraction of Hawthorn Pectin and Its In Vitro Antioxidant and Anti-Glycation Activity" was used for determination. The specific results are shown in Table 5.

[0127] Table 5. Results of anti-glycation activity assays for the examples and comparative examples.

[0128]

[0129] As shown in Table 5, compared with Example 1, Examples 2 and 3 did not show significant changes in the inhibitory activity against AGEs formation after changing the material-liquid ratio and enzyme amount, while the inhibitory activity against AGEs in Comparative Examples 1-3 was significantly reduced. This result indicates that the combined enzymatic hydrolysis of β-glucanase and xylanase is beneficial to the release of active peptides with anti-AGEs formation in leafy grass.

[0130] Furthermore, compared to Example 1, the AGEs inhibitory activity was significantly reduced in Examples 4 and 5 after replacing trypsin with papain and bromelain. This result indicates that, among existing enzymatic digestion combinations, the combined enzymatic digestion using β-glucanase, xylanase, pepsin, and trypsin is the optimal combination for obtaining foliage bioactive peptides with high AGEs inhibitory activity.

[0131] 6. Determination of raw material yield in the examples and comparative examples

[0132] The raw material yields of Examples 1-3 and Comparative Examples 1-5 were calculated using the following formula:

[0133] Yield (%) = M1 / ​​M2 * 100, where: M1 represents the mass (g) of the polypeptide powder obtained after drying the concentrated polypeptide solution obtained by hydrolysis, and M2 represents the mass (g) of the leafy grass raw material.

[0134] The yield results of the raw materials obtained in Examples 1-3 and Comparative Examples 1-5 are shown in Table 6:

[0135] Table 6. Summary of yields for examples and comparative examples

[0136]

[0137] As shown in Table 6, compared with Example 1, the yields of Examples 2 and 3, as well as Comparative Examples 4 and 5, showed no significant changes. However, Comparative Examples 1-3, lacking enzymatic hydrolysis by β-glucanase and / or xylanase, exhibited significantly reduced yields. This result is consistent with the activity results, indicating that β-glucanase or xylanase may facilitate the full enzymatic hydrolysis reaction by promoting the release of cellular proteins and the fluidity of the hydrolysate, thereby increasing the production of active polypeptide fragments and ultimately improving the final yield of active polypeptides from the edible foliage.

[0138] In summary, the method for preparing the active polypeptide of edible leaves provided by the present invention has at least the following technical effects:

[0139] (1) This invention utilizes β-glucanase and xylanase to enzymatically hydrolyze leafweed for the first time. This may improve the fluidity of the subsequent hydrolysate and the enzymatic hydrolysis yield by promoting the release of intracellular proteins and reducing the viscosity of the hydrolysate, respectively. Then, pepsin and trypsin are used to further hydrolyze the hydrolysate to obtain leafweed active polypeptides.

[0140] (2) As can be seen from Example 1 above: when the herbicide peptide is diluted to 10 mg / ml, the pancreatic lipase activity inhibition rate is 55.22%, the anti-glycation activity (anti-AGEs formation) reaches 70.28%, the concentration of γ-aminobutyric acid is 15.69 μg / mL, and the promotion rate for Lactobacillus plantarum BXM2 is 11.28%. Therefore:

[0141] The leafy grass polypeptide prepared by the method of the present invention has lipid-lowering activity, anti-glycation activity (anti-AGEs formation), sleep aid and probiotic functions. It can be used as a raw material to exert the effects of lipid-lowering, regulating intestinal health, sleep aid and preventing skin aging.

[0142] Specifically, by inhibiting the activity of pancreatic lipase, it can effectively suppress the lipolysis process, reduce the digestion and absorption of lipids in food, and thus achieve the goal of controlling and treating obesity. Based on the high pancreatic lipase inhibitory activity of this herbaceous plant bioactive polypeptide, it can be used as an important raw material for lipid-lowering and weight-loss purposes.

[0143] Excessive free radicals in the human body can interfere with cell metabolism, damage proteins, release inflammatory factors, and accelerate aging. Based on the high antioxidant activity of this herbaceous plant active peptide, it can play a role in disease prevention and anti-aging.

[0144] Advanced glycation end products (AGEs) are products of excessive sugar and protein binding. They can combine with and damage body tissues and cells, accelerating aging (such as dull skin, yellowing, and wrinkles) and leading to many chronic degenerative diseases (such as diabetes and cardiovascular diseases). Based on the high anti-AGEs activity of this edible herb, it can be used for cosmetic purposes or to improve chronic diseases.

[0145] Lactobacillus plantarum has the effect of maintaining normal intestinal function and preventing the growth of pathogenic bacteria. Based on the probiotic effect of the active polypeptide of this herb, it can play a role in regulating intestinal health.

[0146] Gamma-aminobutyric acid (GABA) is a naturally occurring non-protein functional amino acid and an important inhibitory neurotransmitter in the central nervous system, exhibiting beneficial effects in calming the nerves, relieving anxiety, and improving sleep. Based on the presence of GABA in this herbaceous plant bioactive polypeptide, it can also promote sleep and soothe emotions.

[0147] In summary, based on their characteristics, phytoesophageal bioactive peptides can be applied to functional products that include at least one of the following functions:

[0148] (1) It possesses pancreatic lipase inhibitory activity;

[0149] (2) It possesses sleep-aiding activity;

[0150] (3) It possesses anti-glycation activity;

[0151] (4) It possesses antioxidant properties;

[0152] (5) It has beneficial bacteria promoting activity.

[0153] Among them, products with the above-mentioned (1)-(5) functions include, but are not limited to, products for regulating intestinal health, lipid-lowering products, sleep aid products and functional food products for preventing skin aging, and may also be products with other obvious effects based on the functions of (1)-(5); wherein, the functional products include pharmaceuticals.

[0154] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of the present invention can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or the background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an active polypeptide from edible leaves, characterized in that: Includes the following steps: Raw material pretreatment: Crush the leafy grass into powder, then add water and stir to obtain a slurry; First hydrolysis: β-glucanase is added to the slurry for the first hydrolysis. After the hydrolysis is completed, the enzyme is inactivated to obtain the first hydrolysate. Second hydrolysis: Xylanase is added to the first hydrolysate for a second hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the second hydrolysate. Third hydrolysis: Pepsin is added to the second hydrolysate for a third hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the third hydrolysate. Fourth hydrolysis: Trypsin is added to the third hydrolysate for a fourth hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the fourth hydrolysate. The fourth hydrolysate is subjected to solid-liquid separation to obtain a supernatant containing the active peptide of *Gnaphalium affine*. The supernatant is then filtered to retain the active peptide of *Gnaphalium affine*, thus obtaining the active peptide of *Gnaphalium affine*. In the raw material pretreatment step, the mass ratio of the leafy grass to water is 1:(10-20). In the first hydrolysis step, β-glucanase is added to the slurry, the pH of the system is adjusted to 4.4-5.1, and hydrolysis is carried out at 45-53°C for 45-80 min; then the enzyme is inactivated at 85-90°C for 10-20 min to obtain the first hydrolysate. In the second hydrolysis step, xylanase is added to the first hydrolysate to adjust the pH of the system to 3.7-4.2, and hydrolysis is carried out at 45-53°C for 45-80 min; then the enzyme is inactivated at 85-90°C for 10-20 min to obtain the second hydrolysate. In the third hydrolysis step, pepsin is added to the second hydrolysate to adjust the pH of the system to 2.7-3.2, and hydrolysis is carried out at 34-38°C for 100-150 min; then enzyme inactivation treatment is carried out at 85-90°C for 10-20 min to obtain the third hydrolysate. In the fourth hydrolysis step, trypsin is added to the third hydrolysate to adjust the pH of the system to 7.5-8.3, and hydrolysis is carried out at 34-38℃ for 50-70 min; then the enzyme is inactivated at 85-90℃ for 10-20 min to obtain the fourth hydrolysate. Based on the protein content in the edible grass, the amount of β-glucanase added is 200-400 U / g; The amount of xylanase added is 150–300 U / g; The amount of pepsin added is 2000-4000 U / g; The amount of trypsin added is 4000-7000 U / g; The fourth hydrolysate was centrifuged at 4000-6000 rpm for 8-15 min to obtain the supernatant; The pH of the supernatant was adjusted to 5.7–6.1, and then subjected to microfiltration through a membrane with a pore size of 0.22–0.45 μm and ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da. The ultrafiltration peptide solution is concentrated using a 200 Da nanofiltration membrane device and then sterilized to obtain a peptide solution with a molecular weight of 200–3000 Da.

2. The method for preparing the herbaceous plant bioactive polypeptide according to claim 1, characterized in that: The polypeptide solution with a molecular weight of 200-3000 Da is concentrated to a polypeptide mass content of 6%-10%, and the concentrate is then made into powder.

3. A leafy herb bioactive polypeptide, characterized in that: The active polypeptide of the leafwort was prepared using the method described in claim 1 or 2.

4. A functional product, characterized in that: Its components include the leafy herb active polypeptide as described in claim 3; The functional product includes at least one of the following functions: (1) It possesses pancreatic lipase inhibitory activity; (2) It possesses sleep-aiding activity; (3) It possesses anti-glycation activity; (4) It possesses antioxidant properties; (5) It has beneficial bacteria promoting activity.

5. The functional product according to claim 4, characterized in that: The functional products include pharmaceuticals.

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