Lactobacillus mucosae fermentation FJ701 and its application in preparing high-biological-activity angelica fermentation

Abstract

The application belongs to the technical field of microbial fermentation, and particularly relates to a fermented lactobacillus mucioparius FJ701 and application of the fermented lactobacillus mucioparius FJ701 in preparation of high-biological-activity angelica fermentation product. The angelica fermentation product prepared by fermentation of the fermented lactobacillus mucioparius FJ701 has high content of dihydroferulic acid, butenyl phthalide, polyphenol and flavone, and in vitro simulation digestion experiments show that the bioavailability of dihydroferulic acid, butenyl phthalide and polyphenol is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a fermentation of Lactobacillus mucilaginosus FJ701 and its application in the preparation of highly bioactive Angelica sinensis ferments. Background Technology

[0002] Angelica sinensis, a traditional Chinese medicinal herb, is sweet, pungent, and warm in nature, and enters the liver, heart, and spleen meridians. It possesses classic effects such as nourishing blood and promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and promoting bowel movements. It is widely used in traditional Chinese medicine clinical practice and in the development of food-medicine homology products. Modern pharmacological research confirms that the core medicinal value of Angelica sinensis stems from its marker components, including volatile oils (ligustilide, butenylphthalide), organic acids (ferulic acid, dihydroferulic acid), and Angelica sinensis polysaccharides. Among these, ligustilide has anti-inflammatory effects and improves microcirculation; ferulic acid has antioxidant and cardiovascular protective effects; and Angelica sinensis polysaccharides have immunomodulatory and hematopoietic-promoting effects.

[0003] However, there are three major technical bottlenecks in the practical application of Angelica sinensis, which seriously restrict its value: First, ligustilide is chemically unstable, volatile and easily decomposed, resulting in serious loss of active ingredients during the production and storage of Angelica sinensis products, making it difficult to maintain stable product efficacy; Second, ferulic acid has low oral bioavailability and limited human absorption efficiency, which limits its efficacy; Third, traditional extraction processes (such as steaming extraction) not only fail to achieve efficient release of active ingredients in Angelica sinensis and have low extraction efficiency, but also the high temperature and other process conditions may destroy some active ingredients, further reducing product quality. Summary of the Invention

[0004] Therefore, the present invention aims to provide a fermentation strain of Lactobacillus mucinus FJ701, which can significantly increase the content of dihydroferruvic acid, butenylphthalide, polyphenols and flavonoids in Angelica sinensis fermentation product, and significantly increase the release of dihydroferruvic acid, butenylphthalide and polyphenols in the intestine.

[0005] The second objective of this invention is to provide an Angelica sinensis fermentation product, which contains high levels of dihydroferruvic acid, butenylphthalide, polyphenols, and flavonoids.

[0006] The third objective of this invention is to provide an Angelica sinensis fermentation product, which results in a high release of dihydroferruvic acid, butenylphthalide, and polyphenols in the intestine, thereby promoting absorption.

[0007] In order to solve the above-mentioned technical problems / achieve the above-mentioned objectives, or at least partially solve the above-mentioned technical problems / achieve the above-mentioned objectives, the first aspect of this application provides a fermenting Lactobacillus mucinus FJ701, which has the accession number CGMCC No.36562 and is deposited at the China General Microbiological Culture Collection Center on November 10, 2025.

[0008] As a second aspect of this application, the use of the aforementioned fermenting Lactobacillus mucinus FJ701 in the preparation of Angelica sinensis ferment or fermented Angelica sinensis is provided.

[0009] As a third aspect of this application, a highly bioactive Angelica sinensis ferment is provided, which is prepared by enzymatic hydrolysis of Angelica sinensis and fermentation with Lactobacillus fermentum FJ701.

[0010] Preferably, the enzyme preparations used for the enzymatic hydrolysis of Angelica sinensis include cellulase and pectinase.

[0011] Preferably, the amount of cellulase added is 0.2-0.6% of the mass of Angelica sinensis powder, and the amount of pectinase added is 0.2-0.6% of the mass of Angelica sinensis powder; more preferably, the amount of cellulase added is 0.5% of the mass of Angelica sinensis powder, and the amount of pectinase added is 0.5% of the mass of Angelica sinensis powder.

[0012] Preferably, the cellulase has an enzyme activity of 10,000-30,000 U / g; the pectinase has an enzyme activity of 3,000-8,000 U / g; more preferably, the cellulase has an enzyme activity of 10,000 U / g and the pectinase has an enzyme activity of 5,000 U / g.

[0013] Preferably, the inoculation amount of the fermenting *Lactobacillus fermentatus* FJ701 strain is 1-4%, and more preferably, the inoculation amount of the fermenting *Lactobacillus fermentatus* FJ701 strain is 2%.

[0014] Preferably, the viable count of the fermenting *Lactobacillus mucinus* FJ701 is 1.0 × 10⁻⁶. 8 CFU / mL or higher.

[0015] A further preferred method for activating the fermenting *Lactobacillus mucinus* FJ701 is as follows: the strain is inoculated into MRS medium and cultured at 35°C for 24 hours, followed by three consecutive subcultures to fully activate the strain to a viable count of 1.0 × 10⁻⁶. 8 CFU / mL or higher.

[0016] A further preferred embodiment of the MRS culture medium is as follows: the culture medium formulation consists of 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2 g / L triammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween-80. The above MRS culture medium is then added to 1 L of pure water and autoclaved at 121°C for 15 min.

[0017] As a fourth aspect of this application, a method for preparing the Angelica sinensis fermentation product described in this application is provided, comprising the following steps: (1) mixing Angelica sinensis and water evenly, adding an enzyme preparation for enzymatic hydrolysis, and then performing enzyme inactivation extraction treatment to obtain Angelica sinensis hydrolysate; (2) inoculating the Angelica sinensis hydrolysate with a fermenting agent for fermentation to obtain Angelica sinensis fermentation product.

[0018] Preferably, in step (1), the ratio of Angelica sinensis to water is 1:(5-20); more preferably, the ratio of Angelica sinensis to water is 1:10.

[0019] Preferably, the enzymatic hydrolysis temperature in step (1) is 45-55℃ and the time is 2-5h; more preferably, the enzymatic hydrolysis temperature is 50℃ and the time is 3h.

[0020] Preferably, the enzyme inactivation extraction temperature in step (1) is 110-130℃ and the time is 10-30 min; more preferably, the enzyme inactivation extraction temperature is 115℃ and the time is 15 min.

[0021] Preferably, the fermentation temperature in step (2) is 30-40℃ and the time is 36-72h; more preferably, the fermentation temperature is 37℃ and the time is 48h.

[0022] As a fifth aspect of this application, the application of the Angelica sinensis fermentation product described in this application or the Angelica sinensis fermentation product prepared by the preparation method described in this application in the preparation of products that promote absorption is provided.

[0023] As a sixth aspect of this application, a microbial preparation is provided, the active ingredient of which includes the aforementioned *Lactobacillus fermentans* FJ701.

[0024] Preferably, the microbial preparation further includes adjuvants for maintaining the preservation of microorganisms.

[0025] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0026] 1. This invention provides a fermentation method using *Lactobacillus mucinus* FJ701, which converts the chemically unstable ligustilide in *Angelica sinensis* into butenylphthalide, which has higher stability and comparable activity, and converts ferulic acid, which has low bioavailability, into dihydroferrulic acid, which has a higher absorption rate. This significantly increases the content of dihydroferrulic acid, butenylphthalide, polyphenols, and flavonoids in the fermented *Angelica sinensis* product, and promotes the release and absorption of dihydroferrulic acid, butenylphthalide, and polyphenols in the intestine.

[0027] 2. This invention provides an Angelica sinensis ferment, which is prepared by enzymatic hydrolysis with cellulase and pectinase followed by fermentation with Lactobacillus fermentum FJ701. The obtained Angelica sinensis ferment has high content of dihydroferruvic acid, butenylphthalide, polyphenols, and flavonoids. Among them, dihydroferruvic acid is crucial for improving the ovarian microenvironment and resisting oxidative stress; butenylphthalide has antispasmodic and circulation-improving activities; polyphenols and flavonoids are powerful natural antioxidants with good potential for scavenging free radicals and reducing oxidative damage, providing a material basis for directly protecting ovarian function. It can be applied to products with antioxidant, antispasmodic, circulation-improving, and ovarian-protecting properties.

[0028] 3. This invention provides a fermented Angelica sinensis product. In vitro digestion experiments have confirmed that the fermented Angelica sinensis product has high release levels of dihydroferruvic acid, butenylphthalide, and polyphenols, which are easily absorbed by the human body and exert antioxidant effects.

[0029] 4. This invention provides a method for preparing Angelica sinensis fermented products, which uses liquid fermentation technology. This method is simple and suitable for industrial production.

[0030] Instructions for the preservation of microbial strains:

[0031] Fermenting Lactobacillus mucilaginosus FJ701, Latin name Limosilactobacillus fermentum It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 10, 2025, with accession number CGMCC No. 36562, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0032] Figure 1 The graph shows the release amounts of dihydroferruvic acid, ferulic acid, butenylphthalide, and ligustilide from the in vitro digestion of Angelica sinensis water extract CK, Angelica sinensis fermented by Lactobacillus plantarum SS04, Angelica sinensis fermented by Lactobacillus mucilaginosus ZJP023, Angelica sinensis fermented by Lactobacillus mucilaginosus FJ701, and Angelica sinensis fermented by Lactobacillus rhamnosus YS08. Different letters in the graph represent significant differences between groups, P < 0.05.

[0033] Figure 2The graph shows the release of flavonoids and polyphenols from the in vitro digestion of Angelica sinensis water extract CK, Angelica sinensis fermented by Lactobacillus plantarum SS04, Angelica sinensis fermented by Lactobacillus mucilaginosus ZJP023, Angelica sinensis fermented by Lactobacillus mucilaginosus FJ701, and Angelica sinensis fermented by Lactobacillus rhamnosus YS08. Different letters in the graph represent significant differences between groups, P < 0.05. Detailed Implementation

[0034] The following non-limiting embodiments are intended to provide a more comprehensive understanding of the invention by those skilled in the art, but do not limit the invention in any way. The following description is merely an exemplary illustration of the scope of protection claimed by the invention. Those skilled in the art can make various changes and modifications to the invention based on the disclosed content, and these should also fall within the scope of protection claimed by the invention. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, the two endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0035] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels. Products from different manufacturers do not have a significant impact on the effectiveness.

[0036] The cellulase was purchased from Xiasheng Food Grade Cellulase Plant Extract Special Enzyme SPE-017L, with an enzyme activity of 10000 U / g; the pectinase was purchased from Xiasheng Food Grade Pectinase SPE-010, with an enzyme activity of 5000 U / g.

[0037] Lactobacillus plantarum FH01, Latin name Lactiplantibacillus plantarum Purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC 1.2469. FH01 is the inventor's self-name, and it is a commercially available strain.

[0038] Lactobacillus plantarum SS04, Latin name Lactiplantibacillus plantarum Purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC 1.2167. SS04 is the inventor's self-name and is a commercially available strain.

[0039] Lactobacillus rhamnosus YS08, Latin name Lactobacillus rhamnosus The strain was purchased from the China Industrial Microbial Culture Collection Center, with strain number CICC 20259. YS08 is the inventor's self-name and is a commercially available strain.

[0040] Lactobacillus rhamnosus LR03, Latin name Lactobacillus rhamnosus The strain was purchased from the China Industrial Microbial Culture Collection Center, with strain number CICC 25096. LR03 is the inventor's self-name and is a commercially available strain.

[0041] Lactobacillus rhamnosus FSH05, Latin name Lactobacillus rhamnosus Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 6001, FSH05 is the inventor's self-name, and is a commercially available strain.

[0042] Lactobacillus fermentum ZJP023, Latin name Limosilactobacillus fermentum The strain was purchased from the China Industrial Microbial Culture Collection Center, with strain number CICC 21827. ZJP023 is the inventor's self-name, and it is a commercially available strain.

[0043] Fermenting Lactobacillus mucinus FJ101, Latin name Limosilactobacillus fermentum Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 22702, FJ101, named by the inventor himself, is a commercially available strain.

[0044] Preparation Example 1: Activation and Cultivation of Microbial Strains

[0045] Activation of lactic acid bacteria: *Lactobacillus plantarum* FH01, *Lactobacillus plantarum* SS04, *Lactobacillus rhamnosus* YS08, *Lactobacillus rhamnosus* LR03, *Lactobacillus rhamnosus* FSH05, *Lactobacillus fermentum* ZJP023, *Lactobacillus fermentum* FJ101, and *Lactobacillus fermentum* FJ701 were inoculated into MRS medium and incubated at 35℃ for 24 h. The culture was subcultured three times to fully activate the bacteria to a viable count of 1.0 × 10⁻⁶. 8 CFU / mL or higher.

[0046] The composition and preparation method of MRS medium are as follows: peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium hydrogen citrate 2 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, and Tween-80 1 g / L. Take the above MRS medium, add 1 L of pure water, and autoclave at 121℃ for 15 min for later use.

[0047] Preparation Example 2: Preparation of Angelica sinensis fermentation product

[0048] This preparation example provides a method for preparing Angelica sinensis fermentation product, including the following steps: (1) Angelica sinensis is ground into powder and passed through a 100-mesh sieve to obtain Angelica sinensis powder; (2) 10 g of Angelica sinensis powder is taken and 100 mL of pure water is added at a solid-liquid ratio of 1:10 (w / v). 0.5% (relative to the mass of Angelica sinensis powder) of cellulase and 0.5% of pectinase are added to the suspension and placed in a 50℃ water bath shaker for 3 h of enzymatic hydrolysis; after the enzymatic hydrolysis is completed, it is sterilized by high-pressure steam at 115℃ for 15 min and cooled to room temperature to obtain Angelica sinensis enzymatic hydrolysate; (3) On a sterile operating table, activated lactic acid bacteria solution is inoculated into the Angelica sinensis enzymatic hydrolysate at an inoculation rate of 2% (where 2% is a volume ratio, for example, if the fermentation volume is 100 mL, 2 mL of bacterial solution is added), and thoroughly mixed. After sealing, it is placed in a 37℃ constant temperature incubator for 48 h of fermentation. After the fermentation is completed, it is sterilized by high-pressure steam at 115℃ for 15 min, cooled to room temperature, and freeze-dried to obtain Angelica sinensis fermentation product.

[0049] During inoculation, ensure a consistent number of viable bacteria in the bacterial solution. Specific lactic acid bacteria strains for inoculation are shown in Table 1. Comparative Example 1 in Table 1 represents the uninoculated strain, replaced by sterile water.

[0050] Table 1. Groups and corresponding lactic acid bacteria inoculation solutions

[0051]

[0052] Preparation Example 3: Angelica sinensis water extract

[0053] Take 10g of Angelica sinensis powder, add 100mL of pure water at a solid-liquid ratio of 1:10 (w / v), mix well, and extract at 50℃ for 3h. Then autoclave at 115℃ for 15min, cool to room temperature, and obtain the Angelica sinensis water extract mixture; on a sterile operating table, inoculate with 2% (v / v) sterile water, mix well, seal, and incubate at 37℃ for 48h. After the incubation, autoclave at 115℃ for 15min, cool to room temperature, and freeze-dry to obtain the Angelica sinensis water extract.

[0054] Experimental Example 1: Determination of the content of substances in Angelica sinensis fermentation products

[0055] 1. Determination of the content of dihydroferruvic acid, ferulic acid, butenylphthalide, and ligustilide.

[0056] 1.1 Preparation of Standard Curve

[0057] 1.1.1 Standards: Dihydroferulic acid (CAS: 1135-23-5, HPLC ≥ 98%), ferulic acid (CAS: 1135-24-6, HPLC ≥ 98%), Butenylphthalide (CAS: 551-08-6, HPLC ≥ 98%), Ligusticolone (CAS: 4431-01-0, HPLC ≥ 98%)

[0058] 1.1.2 Detection Method

[0059] Precisely prepare standard solutions of dihydroferruvic acid, ferulic acid, butenylphthalide, and ligustilide at concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg / mL. HPLC detection conditions: Column: C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: Phase A 0.1% phosphoric acid aqueous solution, Phase B acetonitrile; Column temperature: 30℃; Injection volume: 10 μL; Detection wavelength: 210 nm; Flow rate: 1 mL / min. Elution conditions: Gradient elution: 0–5 min, 20%–25% B; 5–15 min, 25%–40% B; 15–20 min, 40%–50% B; 20–30 min, 50%–70% B; 30–45 min, 70%–20% B; 45–50 min, 20% B.

[0060] Under the above liquid chromatography conditions, the standards were injected and detected, and the standard curve was obtained based on the sample concentration and the detection peak area.

[0061] The standard curves obtained are as follows:

[0062] Dihydroferulic acid standard curve: y=34061114x+435621.67, peak time around 10.239; Ferulic acid standard curve: y=37351720.14x-236565.57, peak time around 11.36; Butenylphthalide standard curve: y=19246436.86x-351939.76, peak time around 33.192; Ligusticolone standard curve: y=33798826.29x-365054.81, peak time around 33.477.

[0063] 1.2 Detection of the sample to be tested

[0064] Samples to be tested: Angelica ferment and Angelica water extract were extracted with 70% ethanol and used as samples to be tested.

[0065] 1 mL of the sample to be tested was drawn up using a 1 mL syringe and filtered through a 0.45 μm filter membrane. The contents of dihydroferruvic acid, ferulic acid, butenylphthalide, and ligustilide were determined by HPLC. The contents of dihydroferruvic acid, ferulic acid, butenylphthalide, and ligustilide in the sample were determined according to the above HPLC detection conditions.

[0066] 1.3 Results Analysis

[0067] Table 2. Contents of dihydroferruvic acid, ferulic acid, butenylphthalide, and ligustilide in Angelica sinensis fermentation products of each group.

[0068]

[0069] Note: Different letters in the same column represent significant differences, P < 0.05.

[0070] Angelica sinensis contains various active ingredients, among which volatile oils, organic acids, and polysaccharides are the main active components. Volatile oils include ligustilide and butenylphthalide, while organic acids include ferulic acid and dihydroferulic acid. However, ligustilide is unstable, and ferulic acid has low bioavailability. As shown in Table 2, the contents of ferulic acid and ligustilide in the aqueous extract and enzymatic hydrolysate of Angelica sinensis are higher than those in the lactic acid bacteria fermentation product. After lactic acid bacteria fermentation, the contents of ligustilide and ferulic acid decrease, while the contents of butenylphthalide and dihydroferulic acid increase. This indicates that lactic acid bacteria can convert the unstable ligustilide in Angelica sinensis into the stable and equally active butenylphthalide, and convert ferulic acid into the more bioavailable dihydroferulic acid.

[0071] Among all the Angelica sinensis fermentation products obtained by fermentation strains, the Angelica sinensis fermentation product obtained by fermentation with Lactobacillus myxoides FJ701 showed a significant increase in dihydroferruvic acid content compared to the water extract and enzymatic hydrolysate of Angelica sinensis (0.14 mg / g), reaching 1.49 mg / g, which is 10.6 times higher than that of the water extract and enzymatic hydrolysate. At the same time, the ferulic acid content decreased the most. This confirms the biotransformation capacity of Lactobacillus myxoides FJ701.

[0072] Dihydroferulic acid, a reduced form of ferulic acid, exhibits significantly higher oral bioavailability and in vivo antioxidant activity compared to ferulic acid. This means the body can absorb and utilize this key active ingredient more efficiently, which is crucial for improving the ovarian microenvironment and combating oxidative stress.

[0073] The content of ligustilide decreased after fermentation, while the content of butenylphthalide increased. The highest increase in butenylphthalide content was observed in the fermented product of *Lactobacillus myxitis* FJ701, reaching 16.98 mg / g, which was generally higher than that of the aqueous extract of *Angelica sinensis* (11.36 mg / g). Ligustilide is extremely unstable, easily volatile and decomposed. Under the action of microorganisms, it is converted into stable butenylphthalide, which not only retains the antispasmodic and circulation-improving activities of ligustilide, but also enhances some of its activities, while maintaining chemical stability. This solves the industry problem of easy loss of active ingredients in *Angelica sinensis* products during production and storage, ensuring the stability and consistency of product efficacy.

[0074] 2. Polyphenol content determination

[0075] Samples to be tested: The Angelica sinensis fermentation products obtained in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 4 and Comparative Example 8 were extracted with 70% ethanol and used as samples to be tested.

[0076] Take 10 mL of Eppendorf tube, add 100 μL of sample and 900 μL of water, then add 1.5 mL of Folin-Ciocalteu and react at room temperature for 5 min. Next, add 1 mL of 20% sodium carbonate and 6.5 mL of water and react at room temperature for 1 hour. Use a pipette to apply 200 μL of the sample. Detect the OD value at 760 nm. Use 0–0.4 mg of gallic acid as a standard polyphenol compound to plot a standard curve for gallic acid. Specific detection results are shown in Table 3.

[0077] Table 3. Results of Polyphenol Content Detection

[0078]

[0079] Note: Different letters in the same column represent significant differences, P < 0.05.

[0080] Results analysis:

[0081] As shown in Table 3, the Angelica sinensis fermented with *Lactobacillus myxoides* FJ701 had the highest polyphenol content, which was significantly higher than that of the enzymatic hydrolysate and other strains. Compared with the enzymatic hydrolysate, the polyphenol content of the Angelica sinensis fermented with *Lactobacillus myxoides* FJ701 was increased by 21.96%, and compared with the Angelica sinensis fermented with *Lactobacillus myxoides* FJ101, the polyphenol content of the Angelica sinensis fermented with *Lactobacillus myxoides* FJ701 was increased by 8%. This indicates that *Lactobacillus myxoides* FJ701 is the preferred strain and can effectively increase the polyphenol content in Angelica sinensis fermented products.

[0082] 3. Determination of flavonoid content

[0083] Samples to be tested: The Angelica sinensis fermentation products obtained in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 4 and Comparative Example 7-8 were extracted with 70% ethanol and used as samples to be tested.

[0084] Using 0-1 mg / mL rutin as the standard flavonoid compound, a standard curve for rutin was plotted, y = 0.238X + 0.0439, R0. 2 =0.9996.

[0085] Take 10 mL of Eppendorf tube, add 200 μL of sample, 2.4 mL of water, and 200 μL of 5% sodium nitrite. React at room temperature for 5 min, then add 200 μL of aluminum nitrate and react at room temperature for 5 min. Finally, add 1 mL of 4% sodium hydroxide and react at room temperature for 15 min. Use a pipette to spot 200 μL of the solution. Measure the OD value at 510 nm. See Table 4 for specific results.

[0086] Table 4. Results of Flavonoid Content Detection

[0087]

[0088] Note: Different letters in the same column represent significant differences, P < 0.05.

[0089] Results analysis:

[0090] As shown in Table 4, the fermented Angelica sinensis product obtained from *Lactobacillus myxoides* FJ701 fermentation had the highest flavonoid content, significantly higher than that of the enzymatic hydrolysate and other fermented products. Specifically, the flavonoid content in the fermented Angelica sinensis product obtained from *Lactobacillus myxoides* FJ701 fermentation was 67.56% higher than that in the enzymatic hydrolysate, indicating that fermentation with *Lactobacillus myxoides* FJ701 can effectively increase the flavonoid content in Angelica sinensis fermentation products and can be considered a preferred strain for increasing the flavonoid content of Angelica sinensis fermentation products. Polyphenols and flavonoids are powerful natural antioxidants. Maintaining and optimizing their content implies that fermented Angelica sinensis has a good potential to scavenge free radicals and reduce oxidative damage, providing a material basis for directly protecting ovarian function.

[0091] In conclusion, Lactobacillus f. J701, as a preferred strain for fermenting Angelica sinensis, can significantly increase the content of butenylphthalide, dihydroferruvic acid, flavonoids and polyphenols in the fermented Angelica sinensis product.

[0092] Experiment Example 2: In vitro simulated digestion experiment of fermented Angelica sinensis

[0093] 1. Experimental samples: Angelica sinensis fermentation product and Angelica sinensis water extract obtained from Example 1, Comparative Examples 3-4, and Comparative Example 7.

[0094] 2. Preparation of simulated digestive fluid:

[0095] Simulated saliva: Weigh 0.021 g KH2PO4, 0.30 g Na2HPO4 and 0.81 g NaCl, dissolve them in an appropriate amount of deionized water, adjust the pH to 6.75, add 0.02 g α-amylase, dissolve and bring the volume to 100 mL for later use.

[0096] Simulated gastric juice: Weigh 5 g of pepsin and 1 g of NaCl, add them to 0.13 mol / L hydrochloric acid and dissolve them completely. Adjust the pH to 2 with 6 mol / L hydrochloric acid and then bring the volume to 500 mL for later use.

[0097] Simulated intestinal fluid: Weigh 3.40 g KH2PO4 and dissolve it in 350 mL deionized water. Add 38.50 mL of 0.20 mol / L NaOH solution and mix thoroughly. Then add 5 g trypsin and 30 g bile salts. After dissolving thoroughly, centrifuge at 4000 r / min for 15 min. Take the supernatant and adjust the pH to 7.6 with 4 mol / L NaOH solution. Then bring the volume to 500 mL for later use.

[0098] 3. In vitro simulated digestion:

[0099] Control group: Weigh 0.5 g of the experimental sample, add 5.00 mL of deionized water and mix well; add pure water in the same volume as the digestion solution and the reagent used to adjust the pH, simulate digestion for 4 h in a constant temperature shaker at 37℃ and 100 r / min in the dark, sonicate at 50℃ for 30 min, centrifuge at 4000 r / min for 15 min, and take the supernatant to obtain the undigested sample.

[0100] Experimental group: 0.50 g of the experimental sample was taken, mixed with 5.00 mL of deionized water, and then 0.50 mL of simulated saliva was added. The mixture was shaken at 100 r / min for 5 min at 37℃, followed by a 5 min boiling water bath to inactivate the enzyme. After cooling, the pH was adjusted to 2.0 with 6 mol / L hydrochloric acid, and 7.5 mL of simulated gastric juice was added. The mixture was shaken at 100 r / min for 120 min at 37℃, followed by a 5 min boiling water bath to inactivate the enzyme. After cooling, the pH was adjusted to 7.6 with 4 mol / L NaOH solution, and 7.5 mL of simulated intestinal juice was added. The mixture was shaken at 100 r / min for 120 min at 37℃, followed by a 5 min boiling water bath to inactivate the enzyme. The mixture was then extracted by sonication at 50℃ for 30 min, centrifuged at 4000 r / min for 15 min, and the supernatant was collected to obtain the intestinal digestion sample.

[0101] The contents of dihydroferulic acid, ferulic acid, butenylphthalide, ligustilide, flavonoids, and polyphenols in the intestinal digested sample were determined according to the content determination method in Experiment Example 1. Specific results are as follows: Figure 1 and Figure 2 As shown.

[0102] Depend on Figure 1 and Figure 2 The results showed that the release of active substances from both the digested Angelica sinensis ferment and the Angelica sinensis water extract was higher than that from the undigested samples. In vitro simulated digestion revealed that the fermented Angelica sinensis sample exhibited a significantly enhanced release of active ingredients compared to the water extract sample.

[0103] Depend on Figure 1The results showed that the contents of butenyl phthalide and dihydroferruvic acid were significantly increased after digestion compared to before digestion. After digestion, the fermented samples exhibited significantly enhanced release of active ingredients, including dihydroferruvic acid and butenyl phthalide, compared to the unfermented samples. Specifically, the Angelica sinensis fermented with *Lactobacillus myxoides* FJ701 showed significantly higher release of dihydroferruvic acid and butenyl phthalide compared to fermented Angelica sinensis from other strains. The Angelica sinensis fermented with *Lactobacillus myxoides* FJ701, after digestion, released 2.0615 mg / g of dihydroferruvic acid, which was 20.27 times that of the digested Angelica sinensis aqueous extract (0.1017 mg / g); the release of butenyl phthalide was 5.8228 mg / g, which was 1.20 times that of the Angelica sinensis aqueous extract (4.8322 mg / g). The results indicate that fermenting Angelica sinensis with Lactobacillus mucinus FJ701, a preferred strain, can significantly increase the release of dihydroferruvic acid and butenylphthalide in the intestine, thus facilitating the absorption of active ingredients.

[0104] Depend on Figure 2 The results showed that, after in vitro simulated digestion, the fermented samples exhibited significantly enhanced release of active ingredients, including flavonoids and polyphenols, compared to the unfermented samples. Specifically, the Angelica sinensis fermented with *Lactobacillus myxoides* FJ701 showed a significantly higher polyphenol content compared to fermented Angelica sinensis from other strains. The flavonoid release from the *Lactobacillus myxoides* FJ701 fermented Angelica sinensis after digestion was 8.4572 mg / g, 2.15 times that of the water extract of Angelica sinensis after digestion (3.9161 mg / g); the polyphenol release was 12.9190 mg / g, 1.38 times that of the water extract of Angelica sinensis (9.3394 mg / g). This indicates that *Lactobacillus myxoides* FJ701, as a preferred strain, can significantly enhance the release of polyphenols in the intestine during fermentation of Angelica sinensis, thus facilitating the absorption of active ingredients.

[0105] In summary, the Angelica sinensis ferment obtained by fermenting Lactobacillus fumarate FJ701 can significantly increase the content of dihydroferruvic acid, butenylphthalide, polyphenols and flavonoids in the ferment. In vitro digestion experiments showed that it can significantly increase the release of dihydroferruvic acid, butenylphthalide and polyphenols, thus promoting human digestion and absorption.

[0106] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentum FJ701, characterized in that, The fermenting Lactobacillus mucinus FJ701 has the accession number CGMCC No.36562 and is deposited at the China General Microbiological Culture Collection Center on November 10, 2025.

2. The application of the fermented Lactobacillus mucinus FJ701 according to claim 1 in the preparation of Angelica sinensis ferment.

3. A highly bioactive Angelica sinensis ferment, characterized in that, Angelica sinensis was prepared by inoculating it with Lactobacillus f. J701 as described in claim 1 after enzymatic hydrolysis.

4. The Angelica sinensis fermented product according to claim 3, characterized in that, Enzyme preparations include cellulase and pectinase.

5. The Angelica sinensis fermented product according to claim 4, characterized in that, The amount of cellulase added is 0.2-0.6% of the weight of Angelica sinensis powder, and the amount of pectinase added is 0.2-0.6% of the weight of Angelica sinensis powder.

6. The method for preparing Angelica sinensis fermented product according to any one of claims 3-5, characterized in that, The steps include: (1) mixing Angelica sinensis and water evenly, adding an enzyme preparation for enzymatic hydrolysis, and then performing enzyme inactivation extraction to obtain Angelica sinensis hydrolysate; (2) inoculating the Angelica sinensis hydrolysate with Lactobacillus fermentum FJ701 as described in claim 1 to obtain Angelica sinensis ferment.

7. The method for preparing Angelica sinensis fermented product according to claim 6, characterized in that, The ratio of Angelica sinensis to water is 1:(5-20).

8. The method for preparing Angelica sinensis fermented product according to claim 6, characterized in that, The inoculum size of the fermenting *Lactobacillus mucinus* FJ701 is 1-4%. The viable count of the fermenting *Lactobacillus mucinus* FJ701 was 1.0 × 10⁻⁶. 8 CFU / mL or higher.

9. The method for preparing Angelica sinensis fermented product according to claim 6, characterized in that, The enzymatic hydrolysis temperature is 45-55℃, and the time is 2-5h; the enzyme inactivation extraction temperature is 110-130℃, and the time is 10-30min; the fermentation temperature is 30-40℃, and the time is 36-72h.

10. The use of the Angelica sinensis fermented product according to any one of claims 3-5 or the Angelica sinensis fermented product obtained by the preparation method according to any one of claims 6-9 in the preparation of Angelica sinensis functional products.

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