Ophiopogon japonicus powder for relieving anxiety and improving sleep as well as preparation method and application of ophiopogon japonicus powder

Abstract

The invention discloses radix ophiopogonis powder for relieving anxiety and improving sleep. The radix ophiopogonis powder is prepared by carrying out fermentation on radix ophiopogonis through plant lactobacillus GDMCC No. 64374 and carrying out enzymolysis treatment through pullulanase. The invention further discloses a preparation method and application of the radix ophiopogonis powder. The saponin content of the radix ophiopogonis powder is obviously increased. The radix ophiopogonis powder can be prepared into products in various forms, has the effects of relieving anxiety mood and improving sleep, is high in bioavailability and free of toxic and side effects, and can be eaten for a long time. Compared with the traditional radix ophiopogonis powder, the radix ophiopogonis powder disclosed by the invention is relatively low in safety risk and more obvious in effects of relieving anxiety mood and improving sleep.

Description

Technical Field

[0001] This invention belongs to the field of food and health product technology, specifically relating to Ophiopogon japonicus powder for relieving anxiety and improving sleep, its preparation method and application. Background Technology

[0002] With the fast pace of modern life, sleep disorders have become a global health problem. Chronic sleep deprivation not only leads to decreased attention and weakened immunity, but is also significantly associated with the risk of chronic diseases such as depression and cardiovascular disease. While current mainstream sleep aids (such as benzodiazepines) are fast-acting, they have side effects such as dependence, drug tolerance, and liver and kidney damage. Therefore, developing natural and safe plant-based sleep aids has become a research hotspot in the fields of functional foods and traditional Chinese medicine.

[0003] Ophiopogon japonicus ( Japanese Ophiopogon It is rich in Ophiopogon japonicus saponins, polysaccharides, and flavonoids, and has the effects of nourishing yin and moistening the lungs, clearing the heart and relieving irritability. Traditional Ophiopogon japonicus powder preparation mostly uses water extraction, alcohol extraction, or mechanical pulverization processes, which have significant drawbacks such as loss of effective ingredients, low bioavailability, high safety risks, and limited functionality. Summary of the Invention

[0004] One objective of this invention is to provide a product that can effectively alleviate anxiety and improve sleep disorders, addressing the technical problems mentioned above.

[0005] Another object of the present invention is to provide a method for preparing the product.

[0006] Another object of the present invention is to provide applications of the product.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a powder of Ophiopogon japonicus for relieving anxiety and improving sleep, wherein the powder is obtained by fermentation of Ophiopogon japonicus with Lactobacillus plantarum GDMCC No. 64374 and enzymatic hydrolysis with pullulanase.

[0008] In a preferred embodiment, the saponin content of the Ophiopogon japonicus powder is 0.5%-2%.

[0009] Secondly, the present invention provides a method for preparing the Ophiopogon japonicus powder, which includes the following steps: S1: Obtain fresh Ophiopogon japonicus, freeze-dry it, grind it into powder, and sift it to obtain Ophiopogon japonicus powder; S2: Disperse the Ophiopogon japonicus powder evenly in water and heat to extract, then cool to 37°C to obtain the fermentation substrate; S3: Inoculate with Lactobacillus plantarum seed liquid for fermentation, sterilize after the reaction is complete to obtain Ophiopogon japonicus fermentation broth; S4: Add pullulanase for enzymatic hydrolysis. After the reaction is complete, the enzyme is inactivated to obtain Ophiopogon japonicus hydrolysate. S5: Filter the Ophiopogon japonicus enzymatic hydrolysate, discard the precipitate, collect the supernatant, and spray dry it to obtain the Ophiopogon japonicus powder.

[0010] In a preferred embodiment, the preparation steps of the *Lactobacillus plantarum* seed solution include: (1) Activation: Lactobacillus plantarum was streaked on MRS solid medium and cultured at 35°C for 48 hours under anaerobic conditions; (2) Liquid culture: Pick a single colony from a plate, inoculate it into MRS liquid medium, and culture it under anaerobic conditions at 35°C for 48 hours to obtain bacterial solution; (3) Subculture: Take 10% of the bacterial solution and add it to MRS liquid culture medium. Culture at 35°C for 12 hours under anaerobic conditions to obtain the seed culture of Lactobacillus plantarum.

[0011] In a preferred embodiment, in step S2, the mass ratio of Ophiopogon japonicus powder to water is 1:100 to 5:100. The heating temperature is 70-90℃, more preferably 80℃, and the extraction time is 1-3 hours, more preferably 2 hours.

[0012] In a preferred embodiment, the concentration (OD600) of *Lactobacillus plantarum* in the *Lactobacillus plantarum* seed solution is 1.0-2.0, more preferably 2.0.

[0013] In a preferred embodiment, the volume ratio of Bacillus plantarum seed culture to fermentation substrate is 1:100 to 5:100, more preferably 5:100.

[0014] In a preferred embodiment, the fermentation culture temperature is 33-37℃, more preferably 35℃, and the time is 10-15 hours, more preferably 12 hours.

[0015] In a preferred embodiment, the sterilization process is carried out at a temperature of 100°C for 20-40 minutes, more preferably 30 minutes.

[0016] In a preferred embodiment, in step S4, the amount of pullulanase used is 0.1-0.5% w / w of the Ophiopogon japonicus fermentation broth, more preferably 0.2-0.5% w / w, and most preferably 0.2% w / w.

[0017] In a preferred embodiment, the enzymatic hydrolysis treatment is carried out at a temperature of 55-65°C, more preferably 60°C, with a pH of 4.5-6, more preferably 5, a stirring speed of 150-250 rpm, more preferably 200 rpm, and a time of 5-7 hours, more preferably 6 hours.

[0018] In a preferred embodiment, the enzyme inactivation treatment is carried out at a temperature of 90-100°C, more preferably 90°C, for a time of 10-20 min, more preferably 15 min.

[0019] After fermentation and enzymatic hydrolysis, the resulting Ophiopogon japonicus powder produces many small-molecule polysaccharides, which improves its solubility. The saponin content of the obtained Ophiopogon japonicus powder is 0.5%-2%, which is significantly higher than the 0.1%-0.2% saponin content of traditional water-extracted Ophiopogon japonicus powder.

[0020] Thirdly, the present invention also provides the application of the aforementioned Ophiopogon japonicus powder as a core active ingredient in the preparation of foods, health products, and medicines for relieving anxiety and improving sleep.

[0021] The Ophiopogon japonicus powder, when combined with commonly used excipients in food, health products, and pharmaceuticals, can be prepared into various forms of products. Compared to Ophiopogon japonicus powder obtained through traditional preparation methods, this powder has higher bioavailability, can more effectively relieve anxiety and improve sleep, has lower safety risks, and can be taken by patients for extended periods.

[0022] Compared with the prior art, the present invention has the following advantages: (1) Compared with the Ophiopogon japonicus powder obtained by traditional preparation methods, the saponin content of the Ophiopogon japonicus powder of the present invention is significantly increased.

[0023] (2) Compared with the traditional preparation method, the Ophiopogon japonicus powder of the present invention has higher bioavailability, can effectively relieve anxiety and improve sleep, has lower safety risk, and can be taken by patients for a long time.

[0024] (3) The preparation method provided by this invention is highly operable and easy to standardize and intelligentize. The fermentation-assisted enzymatic hydrolysis method is more environmentally friendly and efficient than traditional extraction and purification methods, and can be scaled up in industrial production.

[0025] To address the aforementioned issues, fermentation-assisted enzymatic hydrolysis technology offers an innovative pathway for the functionalization of Ophiopogon japonicus powder. Targeted transformation is achieved through the synergistic action of specific bacterial strains and bioenzymes, and the room-temperature reaction eliminates the need for chemical solvents, reducing the risk of heavy metal and pesticide residue migration. Therefore, fermentation technology balances efficacy and safety, and this technological breakthrough provides a novel solution for developing sleep health products that combine safety and efficacy. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0027] Unless otherwise specified, all instruments and reagents used in the examples are conventional instruments or reagents in the art and are commercially available products. Unless otherwise specified, all specific experimental operations involved in the text are understandable or known to those skilled in the art based on their common knowledge or conventional technical means, and will not be described in detail here.

[0028] In this embodiment, *Lactobacillus plantarum* ( Lactiplantibacillus plantarum GDMCC No. 64374, selected independently, has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No. 64374, deposit date of February 5, 2024, and deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0029] The preparation steps of Bacillus plantarum seed culture are as follows: (1) Activation: Lactobacillus plantarum GDMCC No.64374 was streaked on MRS solid medium and cultured at 35℃ for 48 hours under anaerobic conditions; (2) Liquid culture: Pick a single colony from a plate, inoculate it into MRS liquid medium, and culture it under anaerobic conditions at 35°C for 48 hours to obtain bacterial solution; (3) Subculture: Take 10% of the bacterial solution and add it to MRS liquid culture medium. Culture at 35°C for 12 hours under anaerobic conditions to obtain the seed culture of Lactobacillus plantarum.

[0030] The formula for MRS solid culture medium is as follows: Dissolve the following components in 1L of distilled water: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.5g magnesium sulfate, 0.25g manganese sulfate, and 15g agar powder. Adjust the pH to 6.2-6.4 and autoclave (101 kPa, 121℃) for 15 minutes.

[0031] The formula for MRS liquid culture medium is as follows: Dissolve the following components in 1L of distilled water: 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.5g magnesium sulfate, and 0.25g manganese sulfate. Adjust the pH to 6.2-6.4 and autoclave (101 kPa, 121°C) for 15 minutes.

[0032] Example 1 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0033] (2) Take 50g of Ophiopogon japonicus powder, add it to 1000g of water, and extract with hot water (80℃, 2h) to obtain the fermentation substrate.

[0034] (3) Take 50 mL of Bacillus plantarum seed liquid (the concentration of Bacillus plantarum OD600 is 2.0) and inoculate it into 1000 mL of fermentation substrate to obtain a fermentation system. Ferment the fermentation system in a shaker at 35℃ for 12 hours to obtain the fermentation product.

[0035] (4) Sterilize the fermentation product at 100℃ for 30 minutes to inactivate the bacteria.

[0036] (5) Add 0.2% (w / w) pullulanase (Sigma CAS#: 9075-68-7), and incubate at pH=5, 60℃ with constant stirring (200rpm) for 6 hours, followed by enzyme inactivation at 90℃ for 15 minutes.

[0037] (6) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder. Example 2 (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0038] (2) Take 30g of Ophiopogon japonicus powder, add it to 1000g of water, and extract with hot water (70℃, 3h) to obtain the fermentation substrate.

[0039] (3) Take 30 mL of Bacillus plantarum seed liquid (the concentration of Bacillus plantarum OD600 is 2.0) and inoculate it into 1000 mL of fermentation substrate to obtain a fermentation system. Ferment the fermentation system in a shaker at 37℃ for 10 hours to obtain the fermentation product.

[0040] (4) Sterilize the fermentation product at 100℃ for 40 minutes to inactivate the bacteria.

[0041] (5) Add 0.5% (w / w) pullulanase, and incubate at pH=4.5 at 65℃ with stirring (150rpm) for 7 hours, followed by enzyme inactivation at 90℃ for 20 minutes.

[0042] (6) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder.

[0043] Example 3 (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0044] (2) Take 10g of Ophiopogon japonicus powder, add it to 1000g of water, and extract with hot water (90℃, 1h) to obtain the fermentation substrate.

[0045] (3) Take 10 mL of Bacillus plantarum seed liquid (the concentration of Bacillus plantarum OD600 is 1.0) and inoculate it into 1000 mL of fermentation substrate to obtain a fermentation system. Ferment the fermentation system in a shaker at 33℃ for 15 hours to obtain the fermentation product.

[0046] (4) Sterilize the fermentation product at 100℃ for 20 minutes to inactivate the bacteria.

[0047] (5) Add 0.1% (w / w) pullulanase, and incubate at pH=6, 55℃ with constant stirring (250rpm) for 5 hours, followed by enzyme inactivation at 100℃ for 10 minutes.

[0048] (6) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder.

[0049] Comparative Example 1 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0050] (2) Take 50g of Ophiopogon japonicus powder, add it to 1000g of water, and extract with hot water (80℃, 2h) to obtain the fermentation substrate.

[0051] (3) Take 50 mL of Bacillus plantarum seed liquid and inoculate it into 1000 mL of fermentation substrate to obtain a fermentation system. Ferment the fermentation system in a shaker at 35°C for 12 hours to obtain the fermentation product.

[0052] (4) Sterilize the fermentation product at 100℃ for 30 minutes to inactivate the bacteria.

[0053] (5) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder. Comparative Example 2 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0054] (2) Take 50g of Ophiopogon japonicus powder, add it to 1000g of water, and extract with hot water (80℃, 2h).

[0055] (3) Add 0.2% (w / w) pullulanase (Sigma CAS#: 9075-68-7), and incubate at pH=5, 60℃ with constant stirring (200rpm) for 6 hours, followed by enzyme inactivation at 90℃ for 15 minutes.

[0056] (4) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder. Comparative Example 3 Prepare Ophiopogon japonicus powder according to the following steps: (1) Take fresh Ophiopogon japonicus, freeze dry, grind into powder, and sieve (20 mesh) to obtain Ophiopogon japonicus powder.

[0057] (2) Take 50g of Ophiopogon japonicus powder, add it to 1000g of water, and extract with hot water (80℃, 2h).

[0058] (3) Filter, discard the precipitate, collect the supernatant, and spray dry to obtain Ophiopogon japonicus powder. Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that commercially available Lactobacillus plantarum (purchased from the China General Microbiological Culture Collection Center, CGMCC No. 1.16089) was used instead of Lactobacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the fermentation and enzymatic hydrolysis conditions were the same.

[0059] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that commercially available Lactobacillus acidophilus (purchased from the China General Microbiological Culture Collection Center, CGMCC No. 1.1854) was used instead of Lactobacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the fermentation and enzymatic hydrolysis conditions were the same.

[0060] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that commercially available Lactobacillus rhamnosus (purchased from the China General Microbiological Culture Collection Center, CGMCC No. 1.8882) was used instead of Lactobacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the fermentation and enzymatic hydrolysis conditions were the same.

[0061] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that commercially available Lactobacillus casei (purchased from the China General Microbiological Culture Collection Center, CGMCC No. 1.8727) was used instead of Lactobacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the fermentation and enzymatic hydrolysis conditions were the same.

[0062] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that commercially available common Candida utilis (purchased from the China General Microbiological Culture Collection Center, CGMCC No. 2.3047) was used instead of Bacillus plantarum GDMCC No. 64374 for fermentation. Apart from the different strains, the fermentation and enzymatic hydrolysis conditions were the same.

[0063] Efficacy Experiment 1. Saponin content detection: The saponin content of the saponin powders in Examples 1-3 and Comparative Examples 1-8 was determined by HPLC. The results are shown in Table 1.

[0064] Table 1. Saponin Content

[0065] As shown in Table 1, compared with traditional saponin powder (Comparative Example 3), or fermentation with only *Lactobacillus plantarum* GDMCC No. 64374 (Comparative Example 1), or enzymatic hydrolysis with only pullulanase (Comparative Example 2), the saponin content of *Ophiopogon japonicus* powder obtained in Examples 1-3 of this invention is as high as 0.55-1.98%, which is significantly improved through the synergistic effect of *Lactobacillus plantarum* GDMCC No. 64374 and pullulanase. Compared with ordinary *Lactobacillus plantarum* or other microbial strains, the *Lactobacillus plantarum* GDMCC No. 64374 independently screened in this invention has higher microbial fermentation activity, and the fermentation product contains more saponins.

[0066] 2. Bioavailability test of Ophiopogon japonicus powder and traditional Ophiopogon japonicus powder Experimental methods: (1) Prepare fresh culture medium: DMEM cell culture medium (Gibco, USA): fetal bovine serum (Gibco, USA): penicillin antibiotic (Guangzhou Ruishu Biotechnology Co., Ltd.) = 9:1:0.1. When CaCo-2 (ATCC cell bank) cells have grown and fused to 80-90% confluence, digest the cells, gently pipette to mix, and then count them. Inoculate with 0.5 mL of cell suspension (2 × 10⁻⁶ cells / mL). 5 Pour the contents onto the 12-well Transwell plate filter wells and add 1.5 mL of complete culture medium.

[0067] (2) The culture medium was replaced daily with fresh culture medium to obtain differentiated monolayers. On days 11, 13, 15, 17, 19 and 21, the integrity of the Caco-2 cell monolayer was measured by epithelial resistance (TEER) using a Millicell-ERS electrode.

[0068] (3) When the TEER value reaches 400 Ω / cm on the 21st day 2 At this point, further transport experiments were conducted using a single-layer membrane.

[0069] (4) Wash the filter holder twice with Hanks balanced salt solution (HBSS, pH 6.8, 37°C) to remove residual culture medium. Add 1 mL of substrate (2 mg / mL) dissolved in HBSS to the upper chamber and 1 mL of HBSS (37°C) to the outer chamber of the substrate.

[0070] (5) At 30, 60, 90 and 120 min, 0.6 mL of sample was collected from the lower chamber, and the same volume (0.6 mL) of HBSS was added to the receiving chamber at the same time.

[0071] (6) Liquid phase detection of the content of the sample permeating into the lower chamber at different time periods, and use permeability to evaluate bioavailability.

[0072] The results are shown in Table 2.

[0073] Table 2. Bioavailability Test Results

[0074] The results showed that the bioavailability of the Ophiopogon japonicus powder of the present invention was 9.78%-19.54%, which was significantly higher than that of other comparative examples, indicating that the Ophiopogon japonicus powder of the present invention has higher bioavailability than Ophiopogon japonicus powder fermented by other bacteria.

[0075] 3. RIN-14B cytotoxicity assay Experimental methods: Prepare fresh culture medium. 1640 basal medium (Gibco, USA): fetal bovine serum (Gibco, USA): penicillin antibiotics (Guangzhou Ruishu Biotechnology Co., Ltd.) = 9:1:0.1.

[0076] When RIN-14B cells (rat insulinoma cells, ATCC cell bank) have grown and fused to 80-90% confluence, the cells are digested, gently pipetted to mix, and then counted. The cells are then counted at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 0.1 mL per well in 96-well plates. After 4 hours of cell attachment, the culture supernatant was discarded, and complete culture medium containing different concentrations of Ophiopogon japonicus powder (0.5, 1, 2, 4, 6 mg / mL) was added and the cells were incubated for another 24 hours. Cell viability was then assessed according to the CCK-8 assay kit instructions.

[0077] The results are shown in Table 3.

[0078] Table 3. Results of RIN-14B cytotoxicity assay

[0079] The cytotoxicity test results showed that the maximum safe concentration of fermented Ophiopogon japonicus powder was 4 mg / mL, while the maximum safe concentration of unfermented and unhydrolyzed Ophiopogon japonicus powder was 1 mg / mL. This indicates that fermentation with different strains can improve the biosafety of Ophiopogon japonicus powder, and a concentration of 1 mg / mL will be used uniformly in subsequent experiments.

[0080] 4. Effect of the sample on the release of serotonin (5-HT) in RIN-14B cells 5-HT is an important monoamine neurotransmitter in the central nervous system, often referred to as the "happy hormone," and also participates in sleep-wake regulation. RIN-14B is a rat islet tumor-derived cell line that synthesizes, stores, and secretes 5-HT, with a secretion mechanism similar to that of 5-HTergic neurons in vivo. It can be used to evaluate the anxiety-relieving and sleep-improving effects of samples.

[0081] Experimental Methods: RIN-14B (rat insulinoma cells, ATCC cell bank) cells were grown and fused to 80-90% confluence. The cells were then digested, gently pipetted to mix, and counted. Cells were counted at a rate of 30 × 10⁻⁶. 4 Cells were seeded at a density of 1 cell per well in 24-well plates and allowed to adhere overnight.

[0082] The experiment was divided into a normal group and a sample group. Cells in each group were washed with a washing solution containing fluoxetine and BSA to inhibit the reuptake of 5-HT. After the sample group was treated with the sample solution for 24 hours, the supernatant was taken and the 5-HT content was tested using an ELISA kit.

[0083] The results are shown in Table 4.

[0084] Table 4. Serotonin promotion rate in RIN-14B cells

[0085] The results show that the sample with higher saponin content has a higher rate of promoting 5-HT in cells. The Ophiopogon japonicus powder of the present invention has the best effect, indicating that the content of Ophiopogon japonicus saponins may be related to relieving anxiety and improving sleep.

[0086] 5. Sedative and hypnotic effects of zebrafish on insomnia 120 hpf embryos were selected and set up as blank control group, model control group, positive control group (melatonin, 1 μM) and sample group (2 g / L). The blank control group and model control group were given embryo buffer water, while the positive control group and sample group were given the corresponding concentration of drug solution. The embryos were incubated in a constant temperature incubator at (28.5±0.5)℃ for 1 day.

[0087] Embryos cultured with the drug were selected into 96-well plates according to their groups, and the drug solution was replaced (100 μL per well). The model control group was given the modeling agent (PTZ, 5 mM), while the positive control group and the sample group were given the corresponding concentrations of the modeling agent mixture. The samples were then transferred to a zebrafish behavioral analysis system, and the movement trajectory of zebrafish under dark conditions was recorded using the device's built-in infrared tracking software for 1 hour. The reduction rate of the total movement distance of the zebrafish was used to indicate the sedative-hypnotic effect of the samples.

[0088] The results are shown in Table 5.

[0089] Table 5. Sedative-hypnotic effects of zebrafish on insomnia

[0090] The results show that the Ophiopogon japonicus powder of the present invention has the best sedative and hypnotic effect, which is better than that of the comparative example.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A type of Ophiopogon japonicus powder for relieving anxiety and improving sleep, characterized in that, The Ophiopogon japonicus powder is produced by fermentation of Ophiopogon japonicus with Lactobacillus plantarum GDMCC No. 64374 and enzymatic hydrolysis with pullulanase.

2. The Ophiopogon japonicus powder according to claim 1, characterized in that, The saponin content of the Ophiopogon japonicus powder is 0.5%-2%.

3. A method for preparing Ophiopogon japonicus powder as described in claim 1 or 2, comprising the following steps: S1: Obtain fresh Ophiopogon japonicus, freeze-dry it, grind it into powder, and sift it to obtain Ophiopogon japonicus powder; S2: Disperse the Ophiopogon japonicus powder evenly in water and heat to extract, then cool to 37°C to obtain the fermentation substrate; S3: Inoculate with Lactobacillus plantarum seed liquid for fermentation, sterilize after the reaction is complete to obtain Ophiopogon japonicus fermentation broth; S4: Add pullulanase for enzymatic hydrolysis. After the reaction is complete, the enzyme is inactivated to obtain Ophiopogon japonicus hydrolysate. S5: Filter the Ophiopogon japonicus enzymatic hydrolysate, discard the precipitate, collect the supernatant, and spray dry it to obtain the Ophiopogon japonicus powder.

4. The method according to claim 3, characterized in that, The preparation steps of the *Lactobacillus plantarum* seed solution include: (1) Activation: Lactobacillus plantarum GDMCC No.64374 was streaked on MRS solid medium and cultured at 35℃ for 48 hours under anaerobic conditions; (2) Liquid culture: Pick a single colony from a plate, inoculate it into MRS liquid medium, and culture it under anaerobic conditions at 35°C for 48 hours to obtain bacterial solution; (3) Subculture: Take 10% of the bacterial solution and add it to MRS liquid culture medium. Culture at 35°C for 12 hours under anaerobic conditions to obtain the seed culture of Lactobacillus plantarum.

5. The method according to claim 3, characterized in that, The mass ratio of the Ophiopogon japonicus powder to the water is 1:100 to 5:

100.

6. The method according to claim 3, wherein the volume ratio of the *Lactobacillus plantarum* seed culture to the fermentation substrate is 1:100 to 5:

100.

7. The method according to claim 3, characterized in that, The fermentation culture was carried out at a temperature of 33-37℃ for 10-15 hours.

8. The method according to claim 3, characterized in that, The pullulanase was used at 0.1-0.5% w / w of the Ophiopogon japonicus fermentation broth.

9. The method according to claim 3, characterized in that, The enzymatic hydrolysis temperature is 55-65℃, the pH is 4.5-6, the stirring speed is 150-250 rpm, and the time is 5-7 hours.

10. The use of Ophiopogon japonicus powder as a core active ingredient in the preparation of foods, health products, and pharmaceuticals for relieving anxiety and improving sleep, as described in claim 1 or 2.

Images