Lactobacillus reuteri xs82-5 and application thereof in preparing a food-cum-medicine fermented product with improved liver qi stagnation

Abstract

The application belongs to the technical field of microorganisms, and provides a lactobacillus rhamnosus XS82-5 and application of the lactobacillus rhamnosus XS82-5 in preparation of a medicine-food homologous fermentation product with the function of improving liver qi stagnation. The preservation number of the lactobacillus rhamnosus XS82-5 is CGMCC No.39597. The fermentation product obtained by fermentation of the lactobacillus rhamnosus XS82-5 can increase the weight gain, sugar water consumption ratio, open field test level and vertical movement score of rats, reduce the content of corticosterone (CORT) in the plasma of rats, and improve the content of neurotrophic factor (BDNF), 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE) in the cortex and hippocampus of rats, so as to achieve the function of improving liver qi stagnation from the aspects of behavior and molecule.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Lactobacillus reuteri XS82-5 and its application in the preparation of medicinal and edible fermented products that improve liver qi stagnation. Background Technology

[0002] Liver Qi stagnation, also known as liver Qi obstruction or liver Qi stagnation, is one of the most common syndromes in Traditional Chinese Medicine (TCM) clinical practice. From the perspective of TCM pathogenesis, the liver plays a leading role in the generation and differentiation of emotions, governs the body's Qi, and regulates the generation and flow of emotions. It is a key hub for the body's Qi regulation, and emotional damage first affects the liver. Typical clinical manifestations of liver Qi stagnation include depression, irritability, frequent sighing, chest and lower abdominal distension and pain, epigastric fullness, loss of appetite, insomnia, and dream-disturbed sleep. Women often experience breast tenderness, dysmenorrhea, and menstrual irregularities.

[0003] Prolonged stagnation can generate internal heat, often causing dry mouth, bitter taste, dizziness, eye strain, and restlessness. It can also trigger or worsen anxiety and depression, and increase the incidence of various proliferative and stagnation-related diseases such as breast nodules. If left untreated, long-term stagnation of Qi can lead to liver Qi stagnation and blood stasis, causing the distending pain in the hypochondrium to turn into fixed stabbing pain. It can also affect the spleen and stomach, often resulting in loss of appetite, irregular bowel movements, and significant menstrual cramps and dark menstrual blood with large clots in women, seriously impacting physical and mental health and quality of life.

[0004] Currently, the main methods for treating liver qi stagnation are oral Chinese herbal decoctions and prepared Chinese medicines, as well as acupuncture and massage. However, these methods have drawbacks such as inconvenience in taking them, slow onset of action, and limited suitability for certain groups of people. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a fermented product that is both food and medicine and its preparation method, wherein the fermented product has the effect of improving depression caused by liver qi stagnation.

[0006] The second objective of this invention is to provide a strain of *Lactobacillus reuteri* XS82-5, which can significantly enhance the effect of fermentation products on depression caused by liver qi stagnation when fermenting food-medicine homologous compositions.

[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, as a first aspect of the present invention, a *Lactobacillus reuteri* (…) is provided. Limosilactobacillus reuteri The Lactobacillus reuteri XS82-5, with accession number CGMCC No. 39597, is deposited at the China General Microbiological Culture Collection Center on July 6, 2026.

[0008] As a second aspect of the invention, the use of *Lactobacillus reuteri* XS82-5 in fermenting food-medicine homologous compositions or preparing food-medicine homologous fermented products is provided. In some embodiments of this application, the use of *Lactobacillus reuteri* XS82-5 as the sole fermentation strain in the preparation of fermented products is provided.

[0009] As a third aspect of the present invention, a food-medicine homology fermentation product that improves liver qi stagnation is provided, which is prepared by enzymatic hydrolysis of a food-medicine homology composition and fermentation with Lactobacillus reuteri XS82-5.

[0010] Preferably, the Lactobacillus reuteri XS82-5 has the accession number CGMCC No.39597, is deposited at the China General Microbiological Culture Collection Center, and the deposit date is July 6, 2026.

[0011] Preferably, the inoculum size of *Lactobacillus reuteri* XS82-5 is 5-10%, and the inoculum size is v / v, which is the proportion of the volume of the *Lactobacillus reuteri* XS82-5 bacterial suspension to the total fermentation volume.

[0012] Preferably, the viable count of the *Lactobacillus reuteri* XS82-5 is 1.0 × 10⁻⁶. 11 CFU / mL or higher.

[0013] Preferably, the medicinal and edible composition includes citron, perilla, Ampelopsis japonica leaves, Sophora japonica buds, and double-petaled red rose.

[0014] More preferably, the medicinal and edible composition comprises, by weight, 30-60 parts of citron, 25-50 parts of perilla, 10-20 parts of Ampelopsis japonica leaf, 1-4 parts of Sophora japonica flower bud, and 10-25 parts of double-petaled red rose.

[0015] In a further preferred embodiment, by weight, the following components are present: 40-60 parts of citron, 30-50 parts of perilla, 15-20 parts of Amaryllis leaf, 2-4 parts of Sophora japonica buds, and 15-25 parts of double-petaled red rose.

[0016] Preferably, the enzyme preparations used for enzymatic hydrolysis include cellulase, pectinase, and neutral protease.

[0017] Further preferred, by weight, it includes 5-10 parts cellulase, 3-8 parts pectinase and 0.5-2 parts neutral protease.

[0018] A further preferred embodiment, by weight, includes 6-10 parts cellulase, 5-8 parts pectinase, and 1-2 parts neutral protease.

[0019] Preferably, the cellulase has an enzyme activity of 20,000-40,000 U / g, the pectinase has an enzyme activity of 30,000-50,000 U / g, and the neutral protease has an enzyme activity of 50,000-400,000 U / g.

[0020] In some embodiments of the present invention, the cellulase has an enzyme activity of 20,000 U / g, the pectinase has an enzyme activity of 30,000 U / g, and the neutral protease has an enzyme activity of 200,000 U / g.

[0021] As a fourth aspect of the present invention, a method for preparing the medicinal and edible homologous fermentation product of the present invention is provided, comprising the following steps:

[0022] (1) The citron, sophora japonica and ampelopsis japonica leaves in the food-medicine homology composition were extracted with water at high temperature to obtain an aqueous extract;

[0023] (2) Add double-petaled red rose and perilla to the aqueous extract, mix well, add enzyme preparation for enzymatic hydrolysis, and obtain enzymatic hydrolysate;

[0024] (3) The enzyme hydrolysate is heated to inactivate the enzyme and extract it to obtain an enzyme-inactivated extract;

[0025] (4) Inoculate the enzyme-inactivating extract with Lactobacillus reuteri XS82-5 for fermentation to obtain the fermentation product.

[0026] Preferably, the Lactobacillus reuteri XS82-5 has the accession number CGMCC No. 39597.

[0027] Preferably, the preparation method further includes activating the *Lactobacillus reuteri* XS82-5 before fermentation. In some embodiments of the present invention, the activation method of the *Lactobacillus reuteri* XS82-5 is as follows: the strain is inoculated into MRS medium and cultured at 35°C for 24 hours, and passaged three times consecutively to fully activate the strain to a viable count of 1.0 × 10⁻⁶. 11 CFU / mL or higher.

[0028] 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.

[0029] Preferably, the inoculum size of *Lactobacillus reuteri* XS82-5 is 5%-10%, specifically, the inoculum size is v / v, that is, the proportion of the volume of *Lactobacillus reuteri* XS82-5 to the total fermentation volume.

[0030] Preferably, the ratio of the medicinal and edible composition to water is 1:(10-25); in some embodiments of the present invention, the ratio of the medicinal and edible composition to water is 1:20.

[0031] Preferably, in step (1), the high-temperature water extraction temperature is 80-100℃ and the water extraction time is 10-20min; in some embodiments of the present invention, the high-temperature water extraction temperature is 90℃ and the water extraction time is 15min.

[0032] Preferably, the enzymatic hydrolysis time in step (2) is 60-150 min and the enzymatic hydrolysis temperature is 45-60℃; in some embodiments of the present invention, the enzymatic hydrolysis temperature is 50℃ and the enzymatic hydrolysis time is 120 min.

[0033] Preferably, the temperature for enzyme inactivation extraction in step (3) is 100-120℃ and the time is 20-40 min; in some embodiments of the present invention, the temperature for enzyme inactivation extraction is 110℃ and the time is 30 min.

[0034] Preferably, the fermentation temperature in step (4) is 25-40°C and the fermentation time is 30-50h; in some embodiments of the present invention, the fermentation temperature is 35°C and the time is 40h.

[0035] As a fifth aspect of the present invention, the application of the above-mentioned medicinal and edible fermentation products provided by the present invention or the medicinal and edible fermentation products obtained by the above preparation methods in the preparation of products for improving liver qi stagnation is provided.

[0036] As a sixth aspect of the present invention, a microbial preparation is provided, the active ingredient of which includes *Lactobacillus reuteri* XS82-5.

[0037] Preferably, the microbial preparation further includes an adjuvant for maintaining the preservation of the microorganisms.

[0038] The functions of each raw material in the medicinal and edible homologous fermented product of this invention are as follows:

[0039] Citron is the dried, ripe fruit of the Rutaceae plant Citron or Citronella. It has a pungent, slightly bitter, and sour taste, and is warm in nature. It enters the liver, lung, and spleen meridians and has the effects of soothing the liver and relieving depression, regulating qi and harmonizing the middle jiao, and drying dampness and resolving phlegm.

[0040] Perilla is a plant of the Lamiaceae family. Perilla frutescensThe dried leaves (or with tender branches) of (L.) Britt. are pungent and warm in nature, and enter the lung and spleen meridians. They have the effects of relieving exterior syndromes, dispelling cold, regulating qi, and harmonizing the stomach.

[0041] The leaves of *Amorphophallus pachycarpa* are the leaves of *Amorphophallus pachycarpa*, belonging to the genus *Amorphophallus* in the family Vitaceae. They are sweet and bland in taste, cool in nature, and enter the lung, liver, and stomach meridians. They have the effects of dispelling wind and detoxifying, removing blood stasis and reducing swelling, stopping bleeding and relieving pain.

[0042] Sophora japonica, a legume (family Sophora genus) Sophora japonica L. The dried, immature flower buds of the plant are bitter and slightly cold in nature, and enter the liver and large intestine meridians. They have the effects of cooling the blood and stopping bleeding, and clearing liver fire.

[0043] Double-petaled red roses are deciduous shrubs belonging to the Rosaceae family of the Rosales order. They have a sweet and slightly bitter taste and are warm in nature. They enter the liver and spleen meridians. They have the effects of regulating qi and relieving depression, harmonizing blood, and relieving pain.

[0044] The technical solution of the present invention has the following advantages:

[0045] 1. The Lactobacillus reuteri XS82-5 strain provided by this invention can significantly improve the effect of food-medicine homologous fermentation on depression caused by liver qi stagnation.

[0046] 2. The "Clinical Guide to Medical Cases, Volume Six, Depression" states that "depression leads to qi stagnation, and prolonged qi stagnation inevitably transforms into heat. Heat stagnation depletes body fluids and prevents their flow, disrupting the ascending and descending mechanisms. Initially, it injures the qi aspect, and prolonged stagnation extends to the blood aspect, eventually leading to chronic and severe depression." Specifically, in the early stages of liver qi stagnation, it is simply a stagnation of liver qi, with the qi mechanism failing to flow smoothly, corresponding to mild depression, often manifested as low mood, chest tightness, frequent sighing, and fullness in the hypochondrium. If liver qi stagnation persists and cannot be relieved, it will transform into fire, entering the middle stage of the disease. The internal heat disturbs the mind, resulting in typical symptoms of stagnation and heat, such as irritability, insomnia, bitter taste in the mouth, and dry mouth. As the stagnation and heat continue to deplete yin fluids and obstruct blood circulation, the pathogenic factors penetrate from the qi aspect to the blood aspect, developing into a complex state of deficiency with qi stagnation, blood stasis, and depletion of both liver yin and heart blood in the later stages of the disease, corresponding to intractable severe depression.

[0047] The fermented medicinal and edible products provided by this invention for improving liver qi stagnation use five medicinal and edible raw materials: citron, perilla, Ampelopsis japonica leaf, double-petaled red rose, and Sophora japonica bud. Citron soothes the liver and relieves depression, while perilla soothes the liver and regulates qi, and can also relieve stomach qi rising and stomach qi imbalance; double-petaled red rose regulates qi, invigorates blood and relieves pain, Sophora japonica bud clears liver fire, and Ampelopsis japonica leaf clears liver fire and calms the mind. The above raw materials work together to relieve depression of the liver qi stagnation type, qi stagnation transforming into fire type, or qi stagnation and blood stasis type.

[0048] 3. The food-medicine homology fermentation product for improving liver qi stagnation provided by this invention, through enzymatic hydrolysis with specific enzyme preparations (cellulase + pectinase + neutral protease) and fermentation with a specific strain of Lactobacillus reuteri XS82-5, can release effective substances to the maximum extent, thereby achieving the best effect of the fermentation product in relieving depression.

[0049] 4. The present invention provides a method for preparing a medicinal and edible fermented product that improves liver qi stagnation. It provides a synergistic process combining high-temperature extraction, enzymatic hydrolysis, and fermentation. In this process, citron, sophora japonica buds, and amaryllis leaves are first extracted with water at high temperature, and then fermented with double-petaled red rose and perilla using specific enzymatic hydrolysis and specific bacterial strains. The above method has been verified to significantly improve the effect of the fermented product in relieving depression.

[0050] 5. The medicinal and edible fermented products provided by this invention, which improve liver qi stagnation, can increase the weight gain, sucrose consumption ratio, open-box test level, and vertical movement score of rats. From a behavioral perspective, they can improve the emotional symptoms induced by liver qi stagnation, such as anhedonia, decreased interest, and decreased autonomous exploration behavior. From a molecular perspective, they can reduce the content of corticosterone (CORT) in rat plasma, increase the content of neurotrophic factor (BDNF) in the rat cortex and hippocampus, and increase the levels of serotonin (5-HT), dopamine (DA), and norepinephrine (NE). They can regulate the symptoms of hypothalamic-pituitary-adrenal (HPA) axis hyperfunction and significantly increase the level of monoamines to improve depression caused by liver qi stagnation.

[0051] Preservation instructions:

[0052] Biological material: Lactobacillus reuteri XS82-5;

[0053] Accession number: CGMCC No. 39597;

[0054] Classification and nomenclature: Lactobacillus reuteri ( Limosilactobacillus reuteri );

[0055] Deposit date: July 6, 2026;

[0056] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0057] Abbreviation of depositary institution: CGMCC;

[0058] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0059] Figure 1The graph shows the 5-HT content in the cortex and hippocampus of rats in the normal group, model group, positive control group (fluoxetine group), Examples 1-4, and Comparative Examples 1-10. Compared with the model group, in Examples 1-4 and the normal group, △ represents P<0.05, △△ represents P<0.01, and △△△ represents P<0.001; compared with Example 1, in the normal group, Comparative Examples 1-10, and the positive control group (fluoxetine group), # represents P<0.05, ## represents P<0.01, and ### represents P<0.001.

[0060] Figure 2 The graph shows the DA content in the cortex and hippocampus of rats in the normal group, model group, positive control group (fluoxetine group), Examples 1-4, and Comparative Examples 1-10. Compared with the model group, in Examples 1-4 and the normal group, △ represents P<0.05, △△ represents P<0.01, and △△△ represents P<0.001; compared with Example 1, in the normal group, Comparative Examples 1-10, and the positive control group (fluoxetine group), # represents P<0.05, ## represents P<0.01, and ### represents P<0.001.

[0061] Figure 3 The graph shows the NE content in the cortex and hippocampus of rats in the normal group, model group, positive control group (fluoxetine group), Examples 1-4, and Comparative Examples 1-10. Compared with the model group, in Examples 1-4 and the normal group, △ represents P<0.05, △△ represents P<0.01, and △△△ represents P<0.001; compared with Example 1, in the normal group, Comparative Examples 1-10, and the positive control group (fluoxetine group), # represents P<0.05, ## represents P<0.01, and ### represents P<0.001. Detailed Implementation

[0062] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0063] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both 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 one of ordinary skill in the art to which this invention pertains.

[0064] 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.

[0065] Lactobacillus plantarum YSH08, Lactiplantibacillus plantarum It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36182 and has been published in Chinese patent CN121736957A.

[0066] Lactobacillus reuteri XS82, Limosilactobacillus reuteri It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36181 and has been published in Chinese patent CN121736956A.

[0067] The leaves of *Ampelopsis grossedentata* were purchased from Bozhou Qianzheng Pharmaceutical Co., Ltd.; citron, perilla, double-petaled red rose, sophora japonica buds, bitter orange blossom, and Buddha's hand were purchased from Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd.; cellulase and pectinase were purchased from Shandong Longket Enzyme Preparation Co., Ltd.; and neutral protease was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0068] Preparation Example 1: Activation and Cultivation of Microbial Strains

[0069] Activation of lactic acid bacteria:

[0070] Lactobacillus reuteri XS82-5, Lactobacillus plantarum YSH08, and Lactobacillus reuteri XS82 were inoculated into MRS medium and cultured at 35℃ for 24 h. The culture was then passaged three times to fully activate the strains to a viable count of 1.0 × 10⁻⁶. 11 CFU / mL.

[0071] 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.

[0072] Example 1

[0073] This embodiment provides a medicinal and edible fermented product for improving liver qi stagnation. The medicinal and edible composition includes 50g of citron, 3g of sophora japonica buds, 18g of Ampelopsis japonica leaves, 20g of double-petaled red rose, and 40g of perilla. Its preparation method includes the following steps:

[0074] (1) The medicinal and edible raw materials, citron, sophora japonica buds, amaryllis leaves, double-petaled red rose, and perilla, were pulverized and passed through a 200-mesh sieve to obtain powder; (2) 50g of citron powder, 3g of sophora japonica bud powder, 18g of amaryllis leaf powder, and 2620g of water were mixed and extracted at 90℃ for 15min to obtain an aqueous extract; (3) 20g of double-petaled red rose powder and 40g of perilla powder were added to the aqueous extract and mixed evenly to obtain a mixed solution; Add sodium bicarbonate to adjust the pH to 5.6, then add 8g of cellulase, 6g of pectinase and 1.5g of neutral protease and enzymatically hydrolyze at 50℃ for 120min to obtain the enzymatic hydrolysate; (3) heat the enzymatic hydrolysate to 110℃ and inactivate the enzyme for 30min to obtain the enzyme-inactivated extract; (4) after cooling the enzyme-inactivated extract to 35℃, inoculate 8% Lactobacillus reuteri XS82-5 (Preparation Example 1) and ferment at 35℃ for 40h to obtain the fermentation product.

[0075] The amount of *Lactobacillus reuteri* added was 8%, specifically referring to the volume ratio of the bacterial solution obtained in Preparation Example 1 to the fermentation volume being 8%. The enzyme activity of cellulase was 20,000 U / g, the enzyme activity of pectinase was 30,000 U / g, and the enzyme activity of neutral protease was 200,000 U / g.

[0076] Example 2

[0077] This embodiment provides a fermented product of medicinal and edible origin that improves liver qi stagnation. The medicinal and edible ingredients include 40g of citron, 2g of sophora japonica buds, 15g of Ampelopsis japonica leaves, 10g of double-petaled red rose, and 30g of perilla. The preparation method includes the following steps:

[0078] (1) The medicinal and edible raw materials, citron, sophora japonica buds, amaryllis leaves, double-petaled red rose, and perilla, were pulverized and passed through a 200-mesh sieve to obtain powder; (2) 40g of citron powder, 2g of sophora japonica bud powder, 15g of amaryllis leaf powder, and 970g of water were mixed and extracted at 80℃ for 20min to obtain an aqueous extract; (3) 10g of double-petaled red rose powder and 30g of perilla powder were added to the aqueous extract and mixed evenly to obtain a mixed solution; Add sodium bicarbonate to adjust the pH to 5.6, then add 6g of cellulase, 8g of pectinase and 0.5g of neutral protease and enzymatically hydrolyze at 55℃ for 100min to obtain the enzymatic hydrolysate; (4) heat the enzymatic hydrolysate to 100℃ and inactivate the enzyme for 25min to obtain the enzyme-inactivated extract; (5) after cooling the enzyme-inactivated extract to 30℃, inoculate 6% Lactobacillus reuteri XS82-5 (Preparation Example 1) and ferment at 30℃ for 45h to obtain the fermentation product.

[0079] The amount of *Lactobacillus reuteri* added was 6%, specifically referring to the volume ratio of the bacterial solution obtained in Preparation Example 1 to the fermentation volume being 6%. The enzyme activity of cellulase was 20,000 U / g, the enzyme activity of pectinase was 30,000 U / g, and the enzyme activity of neutral protease was 200,000 U / g.

[0080] Example 3

[0081] This embodiment provides a fermented product of medicinal and edible origin that improves liver qi stagnation. The medicinal and edible ingredients include 30g of citron, 4g of sophora japonica buds, 10g of Ampelopsis japonica leaves, 15g of double-petaled red rose, and 25g of perilla. The preparation method includes the following steps:

[0082] (1) The medicinal and edible raw materials, citron, sophora japonica buds, amaryllis leaves, double-petaled red rose, and perilla, were pulverized and passed through a 200-mesh sieve to obtain powder; (2) 30g of citron powder, 4g of sophora japonica bud powder, 10g of amaryllis leaf powder, and 2100g of water were mixed and extracted at 95℃ for 18min to obtain an aqueous extract; (3) 15g of double-petaled red rose powder and 25g of perilla powder were added to the aqueous extract and mixed evenly to obtain a mixed solution; Add sodium bicarbonate to adjust the pH to 5.6, then add 5g of cellulase, 5g of pectinase and 1.0g of neutral protease and enzymatically hydrolyze at 45℃ for 150min to obtain the enzymatic hydrolysate; (4) heat the enzymatic hydrolysate to 105℃ and inactivate the enzyme for 40min to obtain the enzyme-inactivated extract; (5) after cooling the enzyme-inactivated extract to 25℃, inoculate 5% Lactobacillus reuteri XS82-5 (Preparation Example 1) and ferment at 25℃ for 50h to obtain the fermentation product.

[0083] The amount of *Lactobacillus reuteri* added was 5%, specifically referring to the volume ratio of the bacterial solution obtained in Preparation Example 1 to the fermentation volume being 5%. The enzyme activity of cellulase was 20,000 U / g, the enzyme activity of pectinase was 30,000 U / g, and the enzyme activity of neutral protease was 200,000 U / g.

[0084] Example 4

[0085] This embodiment provides a fermented product of medicinal and edible origin that improves liver qi stagnation. The medicinal and edible ingredients include 60g of citron, 1g of sophora japonica buds, 20g of Ampelopsis japonica leaves, 25g of double-petaled red rose, and 50g of perilla. The preparation method includes the following steps:

[0086] (1) The medicinal and edible raw materials, citron, sophora japonica buds, amaryllis leaves, double-petaled red rose, and perilla, were pulverized and passed through a 200-mesh sieve to obtain powder; (2) 60g of citron powder, 1g of sophora japonica bud powder, 20g of amaryllis leaf powder, and 2808g of water were mixed and extracted at 100℃ for 10min to obtain an aqueous extract; (3) 25g of double-petaled red rose powder and 50g of perilla powder were added to the aqueous extract and mixed evenly to obtain a mixed solution; Add sodium bicarbonate to adjust the pH to 5.6, then add 10g of cellulase, 3g of pectinase and 2.0g of neutral protease and enzymatically hydrolyze at 60℃ for 60min to obtain the enzymatic hydrolysate; (4) heat the enzymatic hydrolysate to 120℃ and inactivate the enzyme for 20min to obtain the enzyme-inactivated extract; (5) after cooling the enzyme-inactivated extract to 40℃, inoculate with 10% Lactobacillus reuteri XS82-5 (Preparation Example 1) and ferment at 40℃ for 30h to obtain the fermentation product.

[0087] The amount of *Lactobacillus reuteri* added was 10%, specifically referring to the volume ratio of the bacterial solution obtained in Preparation Example 1 to the fermentation volume being 10%. The enzyme activity of cellulase was 20,000 U / g, the enzyme activity of pectinase was 30,000 U / g, and the enzyme activity of neutral protease was 200,000 U / g.

[0088] Comparative Example 1

[0089] This comparative example provides a medicinal and edible fermented product that improves liver qi stagnation. The only difference between this example and Example 1 is that double-petaled red roses are replaced with equal amounts of double-petaled red roses and bitter orange blossoms, and the weight ratio of double-petaled red roses to bitter orange blossoms is 1:1. Specifically, the medicinal and edible raw materials include 50g of citron, 40g of perilla, 18g of Ampelopsis japonica leaves, 3g of Sophora japonica buds, 10g of double-petaled red roses, and 10g of bitter orange blossoms. The remaining ingredients and preparation methods are the same as in Example 1.

[0090] Comparative Example 2

[0091] This comparative example provides a medicinal and edible fermented product that improves liver qi stagnation. The only difference between this example and Example 1 is the strain. Specifically, Lactobacillus reuteri XS82-5 is replaced with Lactobacillus reuteri XS82. The remaining components and preparation methods are the same as in Example 1.

[0092] Comparative Example 3

[0093] This comparative example provides a medicinal and edible fermented product that improves liver qi stagnation. The only difference between this example and Example 1 is the strain of bacteria. Specifically, Lactobacillus reuteri XS82-5 is replaced with Lactobacillus plantarum YSH08. The other components and preparation methods are the same as in Example 1.

[0094] Comparative Example 4

[0095] This comparative example provides a food-medicine homologous fermented product for improving liver qi stagnation. The only difference between this example and Example 1 is that all raw materials are extracted at high temperatures. The preparation method is as follows:

[0096] (1) The medicinal and edible raw materials, citron, sophora japonica, amaryllis leaf, double-petaled red rose and perilla, were crushed and passed through a 200-mesh sieve to obtain powder; (2) 50g of citron powder, 40g of perilla powder, 3g of sophora japonica powder, 18g of amaryllis leaf powder, 20g of double-petaled red rose powder and 2620g of water were mixed and extracted at 90℃ for 15min to obtain an aqueous extract; (3) Sodium bicarbonate was added to the aqueous extract to adjust the pH to 5.6, and then 8g of cellulase, 6g of pectinase and 1.5g of neutral protease were added and enzymatically hydrolyzed at 50℃ for 120min to obtain an enzymatic hydrolysate; (4) The enzymatic hydrolysate was heated to 110℃ and the enzyme was inactivated for 30min to obtain an enzyme-inactivated extract; (5) After the enzyme-inactivated extract was cooled to 35℃, 8% Lactobacillus reuteri XS82-5 (Preparation Example 1) was inoculated and fermented at 35℃ for 40h to obtain a fermentation product.

[0097] Comparative Example 5

[0098] This comparative example provides a fermented product that is both food and medicine for improving liver qi stagnation. The only difference from Example 1 is that the weight ratio of perilla and double-petaled red rose is adjusted to 1:1. Specifically, the food and medicine ingredients include 50g of citron, 30g of perilla, 18g of Ampelopsis japonica leaves, 3g of Sophora japonica buds, and 30g of double-petaled red rose. The remaining ingredients and preparation method are the same as in Example 1.

[0099] Comparative Example 6

[0100] This comparative example provides a medicinal and edible fermented product for improving liver qi stagnation. The only difference from Example 1 is that it does not include Sophora japonica buds, but replaces them with an equal amount of Ampelopsis japonica leaves. Specifically, the medicinal and edible raw materials include 50g of citron, 40g of perilla, 21g of Ampelopsis japonica leaves, and 20g of double-petaled red rose. The remaining ingredients and preparation method are the same as in Example 1.

[0101] Comparative Example 7

[0102] This comparative example provides a medicinal and edible fermented product that improves liver qi stagnation. The only difference between this example and Example 1 is that it does not include Ampelopsis japonica leaves, and instead replaces Ampelopsis japonica leaves with an equal amount of Sophora japonica buds. Specifically, the medicinal and edible raw materials include 50g of Citronella, 40g of Perilla frutescens, 21g of Sophora japonica buds, and 20g of double-petaled red rose. The remaining ingredients and preparation methods are the same as in Example 1.

[0103] Comparative Example 8

[0104] This comparative example provides a medicinal and edible fermented product that improves liver qi stagnation. The only difference from Example 1 is that citron is replaced with Buddha's hand. Specifically, the medicinal and edible ingredients include 50g Buddha's hand, 40g perilla, 3g sophora japonica buds, 18g Ampelopsis japonica leaves, and 20g double-petaled red rose. The remaining ingredients and preparation method are the same as in Example 1. Buddha's hand is sweet and slightly pungent, warm in nature, and enters the liver and stomach meridians, soothing the liver, regulating qi, and relieving stomach pain. Compared to citron, it also has the effect of soothing the liver and regulating qi.

[0105] Comparative Example 9

[0106] This comparative example provides a medicinal and edible fermented product that improves liver qi stagnation. The only difference between this example and Example 1 is that it does not include Sophora japonica buds and replaces them with an equal amount of Citronella. Specifically, the medicinal and edible raw materials include 53g of Citronella, 18g of Ampelopsis japonica leaves, 20g of double-petaled red rose, and 40g of Perilla frutescens. The remaining ingredients and preparation methods are the same as in Example 1.

[0107] Comparative Example 10

[0108] This comparative example provides a medicinal and edible fermented product that improves liver qi stagnation. The only difference between this example and Example 1 is that it does not include perilla and replaces citron with an equal amount of sophora japonica buds. Specifically, the medicinal and edible raw materials include 53g of sophora japonica buds, 18g of Ampelopsis japonica leaves, 20g of double-petaled red rose, and 40g of perilla. The remaining ingredients and preparation methods are the same as in Example 1.

[0109] Experiment Example 1: Rat Experiment

[0110] 1.1 Sample to be tested

[0111] The fermentation products obtained in Examples 1-4 and Comparative Examples 1-10 were freeze-dried under the following conditions: pre-freezing temperature of -50℃ and pre-freezing time of 24h; freezing temperature of -48℃ and drying time of 12h; and vacuum degree of 8Pa, respectively, to obtain the corresponding test samples.

[0112] 1.2 Experimental Reagents

[0113] The plasma corticosterone (CORT) ELISA kit was purchased from Wuhan Huamei Biotechnology Co., Ltd., catalog number CSB-E07014r-IS; the 5-hydroxytryptamine (5-HT) ELISA kit was purchased from Shanghai Lianshuo Biotechnology Co., Ltd., catalog number AE92976Ra; the dopamine (DA) ELISA kit was purchased from Shanghai Lianshuo Biotechnology Co., Ltd., catalog number AE91019Ra; and the norepinephrine (NE) ELISA kit was purchased from Shanghai Lianshuo Biotechnology Co., Ltd., catalog number AE98983Ra. Fluoxetine capsules were purchased from Eli Lilly Suzhou Pharmaceutical Co., Ltd., specification 20mg / capsule, approval number: HJ20160501HJ20181215.

[0114] 1.3 Laboratory Animals

[0115] SPF-grade male SD rats, weighing 180-220g, were kept at (22±2)℃, with a humidity of (50±10)% and natural light, and with free access to food and water.

[0116] 1.4 Experimental Grouping

[0117] SD rats were acclimatized for 7 days. Rats with similar horizontal and vertical movement scores after the open-box test were selected and randomly divided into a normal group, a model group, a positive control group (fluoxetine group), Examples 1-4, and Comparative Examples 1-10, with 10 rats in each group. The normal group consisted of 5 rats per cage, routinely housed with free access to food and water. The other groups were isolated and subjected to 5 weeks of chronic, mild, and unpredictable stress to establish the rat model. Simultaneously, the normal group and the model group were administered an equal volume of pure water by gavage. Examples 1-4, Comparative Examples 1-10, and the positive control group (fluoxetine group) were administered the drug by gavage at the same time every morning. The positive control group (fluoxetine group) was administered 1.8 mg / kg by gavage daily, while Examples 1-4 and Comparative Examples 1-10 were administered the corresponding sample at 2 g / kg by gavage daily.

[0118] 1.5 Establishment of a model for chronic, mild, and unpredictable stress

[0119] Five rats per cage were kept in the normal group and given free access to water and food. Each rat in the other groups was kept in a single cage and randomly subjected to two different stress stimuli each day. The same stimuli were not to be given consecutively, so that the rats could not predict the occurrence of the stimuli and thus avoid developing adaptation.

[0120] Stress stimuli included restraint for 2 hours, standing on an ice-water platform for 2 hours, noise stimulation (110 dB, 30 min), foot shocks (4 mA, 50 times, 5 s intervals), light stimulation (100,000 l×, 30 min), and tail clamping (approximately 1 cm from the tip of the rat's tail, 2 min). Successful model establishment was characterized by increased body mass, a significant decrease in sucrose consumption ratio, and a marked decrease in open-box test scores.

[0121] 1.6 Index Measurement

[0122] 1.6.1 Rat body weight

[0123] Record the body weight of rats in each group before and after the experiment, and compare the changes in body weight of rats in each group.

[0124] 1.6.2 Sugar Water Preference Experiment

[0125] On days 0 and 35, sucrose consumption experiments were conducted on rats in each group. Three days prior to the formal experiment, rats in each group were acclimatized to sucrose by being housed individually in cages and given two bottles of 1% sucrose solution for 24 hours each, followed by one bottle of 1% sucrose solution and one bottle of pure water for another 24 hours. On day 3, rats were fasted and deprived of water for 24 hours. During the formal experiment, each group of rats was given one bottle of 1% sucrose solution and one bottle of pure water simultaneously. The mass of the solutions was measured before and after the experiment, and the amount of sucrose solution consumed (sucrose consumption) and pure water consumed by each group of rats within 24 hours were observed to calculate the sucrose consumption ratio.

[0126] Sugar solution consumption ratio = Sugar solution consumption / (Sugar solution consumption + Pure water consumption)

[0127] 1.6.3 Open-box experiment

[0128] Behavioral scoring was performed using an open-box test on days 0 and 35. The open-box measured 100cm × 100cm × 50cm, with its bottom divided into 100 (10cm × 10cm) squares, all with the inner surfaces painted black. Each rat was placed sequentially in the central square, and human interference was eliminated. The rats' activities were recorded over 3 minutes. Horizontal movement scores were based on the number of squares the animal traversed; 1 point was awarded for each square traversed (three or more paws stepping in), and 1 point was awarded for every 10 centimeters of walking along a line. Vertical movement scores were based on the number of times the animal stood upright, marked by both feet leaving the bottom; 1 point was awarded regardless of the duration of standing until the feet were placed back down. The open-box was thoroughly cleaned before testing the next rat.

[0129] 1.6.4 Measurement of plasma corticosterone (CORT) levels

[0130] After the behavioral test, about 1.5 mL of blood was collected from the orbital cavity of each rat and placed in a heparin-soaked centrifuge tube. The tube was centrifuged at 8000 r / min for 10 min at 4°C. The supernatant was collected and stored in a -20°C freezer for later testing. The test procedure was strictly performed in accordance with the kit instructions.

[0131] 1.6.5 Measurement of BDNF, 5-HT, DA, and NE levels in the cortex and hippocampus

[0132] After orbital blood collection, rats in each group were euthanized by cervical dislocation, and the whole brain was collected. The cortex and hippocampus were quickly separated on ice, weighed, and placed in cryovials. The cells were then rapidly frozen in liquid nitrogen and transferred to a -80°C freezer for later testing. The levels of BDNF, 5-HT, DA, and NE in the cortex and hippocampus were measured according to the instructions of the rat BDNF, 5-HT, DA, and NE kits.

[0133] 1.7 Experimental Results

[0134] 1.7.1 Body weight results of rats in each group

[0135] Initially, the body weight levels of rats in each group were similar, with no significant difference (P>0.5). Thirty-five days after modeling, rats in the model group experienced decreased appetite due to liver qi stagnation and emotional depression, and their body weight gain was significantly lower than that of the normal group, indicating successful modeling. Rats administered the product via gavage showed significantly higher body weights than the model group, with no significant difference compared to the normal group (P>0.5), demonstrating that the fermented product of this invention can effectively improve the decreased appetite caused by liver qi stagnation.

[0136] 1.7.2 Results of sucrose consumption ratio in each group of rats

[0137] Table 1. Results of sucrose consumption ratio in each group of rats

[0138]

[0139] Note: For Examples 1-4 and the normal group compared with the model group, △ represents P<0.05, △△ represents P<0.01, and △△△ represents P<0.001; for the normal group, comparative examples 1-10 and the positive control group (fluoxetine group) compared with Example 1, # represents P<0.05, ## represents P<0.01, and ### represents P<0.001.

[0140] The core symptom of human depression is anhedonia. The sucrose preference test can reflect the degree of anhedonia loss in rats and their responsiveness to events of interest. Table 1 shows that initially, there was no significant difference in sucrose consumption ratio among the groups (P>0.5). After 35 days of experimentation, the sucrose consumption ratio in the model group was significantly lower than that in the normal group. The rats exhibited anhedonia, decreased interest, and depression due to liver qi stagnation, indicating successful rat model establishment.

[0141] Compared with the model group, the sucrose consumption ratio of rats in Examples 1-4 was significantly increased. There was no significant difference between the positive control group (fluoxetine group), the normal group and Example 1 group. This indicates that the medicinal and edible fermentation products obtained by enzymatic hydrolysis and inoculation with Lactobacillus reuteri XS82-5 of the raw materials selected in this invention, namely "citron, perilla, Ampelopsis japonica leaves, Sophora japonica buds and double-petaled red rose", can better improve the mood symptoms such as anhedonia and decreased interest in depression.

[0142] The difference between Comparative Example 1 and Example 1 is that double-petaled red roses were replaced with double-petaled red roses and bitter orange blossoms. Bitter orange blossoms have the effects of regulating qi, relieving chest tightness, harmonizing the stomach, and stopping vomiting. As shown in Table 1, compared with Example 1, the sugar water consumption ratio of rats in Comparative Example 1 was significantly reduced, indicating that the synergistic effect between double-petaled red roses and citron, perilla, Ampelopsis japonica leaves, and Sophora japonica flowers in this invention can improve the loss of pleasure and decreased interest in rats caused by liver qi stagnation.

[0143] The difference between Comparative Examples 2-3 and Example 1 is that the fermenting agent *Lactobacillus reuteri* XS82-5 was replaced with *Lactobacillus reuteri* XS82 and *Lactobacillus plantarum* YSH08 in sequence. Compared with Example 1, Comparative Examples 2-3 showed a significant decrease in the sugar water consumption ratio of rats (P<0.05), indicating that the choice of fermenting agent is crucial for the effect of food-medicine homologous fermentation in relieving liver stagnation. The strain *Lactobacillus reuteri* XS82-5 selected in this invention can maximally improve the anhedonia and decreased interest induced by liver qi stagnation.

[0144] The difference between Comparative Example 4 and Example 1 is that the raw materials "citron, perilla, Amaryllis leaf, Sophora japonica flower bud and double-petaled red rose" were all extracted at high temperature. Compared with Example 1, Comparative Example 4 showed a decrease in the sugar water consumption ratio of rats, indicating that the raw material extraction method selected in this invention is more suitable for the extraction of active ingredients from each raw material and can better improve the loss of pleasure and decreased interest caused by liver stagnation in rats.

[0145] The difference between Comparative Example 5 and Example 1 is that the weight ratio of perilla and double-petaled red rose was adjusted to 1:1. As shown in Table 1, the ratio of raw materials is equally important for the efficacy of fermentation. The raw materials and the ratio of raw materials provided by this invention are the best at improving the loss of pleasure and decreased interest in rats induced by liver qi stagnation.

[0146] As shown in Table 1, compared with Example 1, Comparative Examples 6-7 and 9-10 showed varying degrees of reduction in sucrose consumption in rats. This indicates that the raw materials selected in this invention, namely "Ampelopsis japonica leaves and Sophora japonica buds, Sophora japonica buds and Citronella", have a synergistic effect, which can maximally improve the rats' loss of pleasure and decreased interest, thereby improving the depressive symptoms caused by liver qi stagnation.

[0147] The difference between Comparative Example 8 and Example 1 is that citron was replaced with a herb that also has the effect of soothing the liver and regulating qi.

[0148] As shown in Table 1, even when the raw materials with the same efficacy are replaced, different effects will be produced due to the synergistic competition between the drugs. The fermentation product obtained by the present invention using the synergistic combination of "citron, perilla, amaryllis leaf, sophora japonica flower and double-petaled red rose" has the best effect on improving the loss of pleasure and decreased interest caused by liver stagnation in rats.

[0149] 1.7.3 Results of open-box experiments in each group of rats

[0150] Table 2 Results of horizontal and vertical movement scores of rats in the open-box experiment for each group

[0151]

[0152] Note: For Examples 1-4 and the normal group compared with the model group, △ represents P<0.05, △△ represents P<0.01, and △△△ represents P<0.001; for the normal group, comparative examples 1-10 and the positive control group (fluoxetine group) compared with Example 1, # represents P<0.05, ## represents P<0.01, and ### represents P<0.001.

[0153] In the open-box experiment, a significant decrease in horizontal and vertical movement scores is an important indicator for assessing depressive-like behavior in rats. Horizontal movement scores reflect the rat's voluntary movement and exploration ability. When rats are depressed, they often exhibit psychomotor inhibition, leading to a significant reduction in the number of squares they stride and the total distance they travel in the open box, as well as a low willingness to move. Vertical movement scores reflect the rat's curiosity and activity level towards the external environment. The loss of interest and anhedonia caused by depression will significantly reduce the number of times the rat stands upright.

[0154] As shown in Table 2, compared with the normal group, the horizontal and vertical movement scores of the rats in the model group were significantly reduced (P<0.01), indicating that the rats' activity decreased, their desire to explore disappeared, and their autonomous exploration behavior was significantly reduced after modeling, indicating that the modeling was successful.

[0155] Compared to the model group, the horizontal and vertical movement scores of rats in Examples 1-4 were significantly increased, indicating that the medicinal and edible fermented products obtained by enzymatic hydrolysis and fermentation with Lactobacillus reuteri XS82-5 can improve behavioral inhibition and depression caused by liver stagnation and restore the rats' autonomous activity and exploration ability.

[0156] Compared with Example 1, Comparative Examples 1 and 5-10 showed varying degrees of reduction in both horizontal and vertical motor scores in each group of rats, indicating that the fermentation product obtained by the synergistic combination of "citron, perilla, Ampelopsis japonica leaf, Sophora japonica flower bud and double-petaled red rose" can better alleviate symptoms of liver qi stagnation and improve depressive behavior.

[0157] Compared with Example 1, Comparative Examples 2-3 showed a significant decrease in both horizontal and vertical movement scores in all groups of rats (P<0.05), indicating that the specific fermentation strain *Lactobacillus reuteri* XS82-5 selected in this invention is the optimal strain. This strain can better improve the exploration ability and curiosity of depressed rats and further improve the symptoms of liver qi stagnation.

[0158] Compared with Example 1, Comparative Example 4 showed a significant decrease in both horizontal and vertical movement scores in rats, indicating that the raw material extraction method affects the efficacy of fermentation products in improving depression. The high-temperature extraction method of citron, perilla, and Ampelopsis japonica leaves provided by this invention is more suitable for the extraction of active ingredients, thereby improving depressive behavior in rats.

[0159] The sugar water consumption test and the open-box test are often used together to assess depressive-like behavior in rats. As shown in Tables 1 and 2, there is a synergistic effect among the five raw materials selected in this invention: citron, perilla, Ampelopsis japonica leaves, Sophora japonica buds and double-petaled red rose. At the same time, the fermented product obtained after fermentation with specific high-temperature water and specific strains is the best at improving depressive behavior in rats.

[0160] 1.7.4 Comparison of plasma CORT levels, cortical and hippocampal BDNF levels in rats from different groups

[0161] Table 3. Results of plasma CORT levels, cortical and hippocampal BDNF levels in rats of each group.

[0162]

[0163] Note: For Examples 1-4 and the normal group compared with the model group, △ represents P<0.05, △△ represents P<0.01, and △△△ represents P<0.001; for the normal group, comparative examples 1-10 and the positive control group (fluoxetine group) compared with Example 1, # represents P<0.05, ## represents P<0.01, and ### represents P<0.001.

[0164] CORT, secreted by the adrenal cortex, is a key downstream product of the HPA axis (hypothalamic-pituitary-adrenal axis) stress pathway. Prolonged emotional suppression and liver qi stagnation can continuously overactivate the HPA axis, prompting the adrenal glands to secrete large amounts of CORT. High concentrations of CORT can damage hippocampal neurons. BDNF, a neurotrophic factor that regulates neuronal growth and plasticity in the brain, is expressed at the highest levels in the cortex and hippocampus, maintaining emotional and cognitive function. In a depressive state, peripheral and intrabrain CORT levels increase while BDNF expression is downregulated, inducing depressive-like behaviors such as anhedonia and reduced activity, and is an important molecular indicator for evaluating depression models.

[0165] As shown in Table 3, compared with the normal group, the CORT content in the plasma of rats in the model group was significantly increased, and the BDNF levels in the cortex and hippocampus were significantly decreased, indicating that the depression model was successfully established.

[0166] Compared with the model group, the CORT content in the plasma of rats in Examples 1-4 was significantly reduced, while the levels of BDNF in the cortex and hippocampus were significantly increased. This indicates that the medicinal and edible fermented products prepared in this invention can significantly downregulate plasma CORT levels to improve the symptoms of hyperfunction of the hypothalamus-pituitary-adrenal (HPA) axis in rats, and upregulate the expression of BDNF in the cortex and hippocampus to promote neuronal growth and differentiation, thereby improving the depressive symptoms caused by liver qi stagnation.

[0167] Compared with Example 1, Comparative Examples 1 and 5-10 showed varying degrees of increase in plasma CORT levels and varying degrees of decrease in cortical and hippocampal BDNF levels in each group of rats. This indicates that the five raw materials selected in this invention, namely "citron, perilla, Ampelopsis japonica leaf, Sophora japonica flower bud, and double-petaled red rose", and the specific ratio of raw materials, can effectively downregulate plasma CORT levels and upregulate the expression of BDNF in the rat cortex and hippocampus to improve depressive symptoms caused by liver stagnation. The combination of the five raw materials has a significant synergistic effect.

[0168] Compared with Example 1, Comparative Examples 2-3 showed a significant increase in CORT levels in the plasma of rats in each group, and a significant decrease in BDNF levels in the cortex and hippocampus. This indicates that different strains have significant differences in the release and transformation effects of active ingredients in the raw materials. The strain *Lactobacillus reuteri* XS82-5 selected in this invention showed the best effect, which can fully promote the release of active components in the raw materials, effectively upregulate the expression of BDNF in the rat cortex and hippocampus, downregulate plasma CORT levels, and alleviate depressive behavior.

[0169] Compared with Example 1, Comparative Example 4 showed a significant increase in CORT content in rat plasma and a significant decrease in BDNF levels in the cortex and hippocampus. This indicates that the present invention, through the rational selection of raw material extraction methods, enables more complete extraction of active ingredients from each raw material, thereby effectively regulating rat HPA axis function, increasing BDNF levels in the brain, and improving depressive-like behavior in rats.

[0170] 1.7.5 Comparison of 5-HT, DA, and NE levels in the cortex and hippocampus of rats in different groups

[0171] Serotonin (5-HT), dopamine (DA), and norepinephrine (NE) are core monoamine neurotransmitters that regulate mood, sleep, and emotional activity. 5-HT primarily regulates mood stability, anxiety, and sleep rhythms; insufficient function can easily lead to symptoms such as depressed mood and anxiety. NE dominates brain arousal levels, energy, and attention; impaired pathway function often manifests as persistent fatigue and mental sluggishness. DA mediates the body's sense of pleasure and reward motivation; impaired function leads to anhedonia and decreased interest. When the synthesis, release, or metabolism of 5-HT, DA, and NE in the central brain tissue is insufficient, it directly induces impaired mood regulation function, which is an important mechanism in the pathogenesis of depression.

[0172] The results for 5-HT, DA, and NE in each group are shown below. Figure 1-3 ,Depend on Figure 1-3 The results showed that, compared with the normal group, the levels of three emotion-related monoamine neurotransmitters, 5-HT, DA, and NE, were significantly reduced in the cortex and hippocampus of rats in the model group, confirming that there was obvious neurotransmitter disorder in the liver stagnation model, which induced depressive-like behavior.

[0173] Compared with the model group, Examples 1-4 all increased the levels of 5-HT, DA, and NE, indicating that the fermentation product provided by the present invention can effectively improve neurotransmitter metabolism disorder caused by liver qi stagnation and further improve depressive symptoms.

[0174] Compared with Example 1, Comparative Examples 1 and 5-10 showed significantly lower levels of 5-HT, DA, and NE, indicating that the medicinal and edible raw materials obtained by screening and the fermentation products obtained by proportioning the present invention can significantly increase the level of monoamines and improve the symptoms of neurotransmitter disorder caused by liver stagnation in rats.

[0175] Compared with Example 1, Comparative Examples 2-3 showed varying degrees of reduction in the levels of 5-HT, DA, and NE, indicating that different strains can affect the efficacy of the fermentation product. Among them, Lactobacillus reuteri XS82-5 was the optimal strain, and the fermentation product obtained from its fermentation had the best effect in improving neurotransmitter disorders caused by liver qi stagnation.

[0176] Compared with Example 1, Comparative Example 4 showed a significant decrease in the levels of 5-HT, DA, and NE in the rat cortex and hippocampus, indicating that the raw material extraction method selected in this invention is more conducive to the release of active ingredients and can better improve the neurotransmitter disorder caused by liver qi stagnation.

[0177] In summary, the food-medicine homologous fermentation products provided by this invention can improve depression-like damage caused by liver qi stagnation from multiple targets at both behavioral and molecular levels.

[0178] At the behavioral level, it can significantly improve the sugar water consumption ratio and horizontal and vertical movement scores of rats, effectively improve depression-like symptoms such as loss of pleasure, behavioral inhibition, and decreased autonomous activity and exploration ability caused by liver stagnation, and restore the body's emotional perception and autonomous behavioral activity.

[0179] At the molecular level, this product can inhibit HPA axis hyperactivity caused by liver stagnation and stress, reduce plasma CORT levels, and correct neuroendocrine disorders; at the same time, it can upregulate BDNF expression in the brain, repair central nervous system plasticity damage; and it can increase the levels of monoamine neurotransmitters such as 5-HT, DA, and NE, thereby improving central mood regulation disorders.

[0180] In summary, this fermentation product can simultaneously regulate the body's behavioral phenotype, neuroendocrine system, neuroplasticity, and monoaminergic nervous system, achieving synergistic internal and external repair of liver-stagnation type depression, with clear intervention targets and comprehensive levels of action.

[0181] 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 type of *Lactobacillus reuteri* ( Limosilactobacillus reuteri XS82-5, characterized in that, The Lactobacillus reuteri XS82-5 has the accession number CGMCC No.39597 and is deposited at the China General Microbiological Culture Collection Center on July 6, 2026.

2. The application of Lactobacillus reuteri XS82-5 according to claim 1 in the fermentation of food-medicine homologous compositions or in the preparation of food-medicine homologous fermented products.

3. A fermented product that is both food and medicine, characterized in that it improves liver qi stagnation, is characterized in that... The food-medicine homology composition was prepared by enzymatic hydrolysis and fermentation with *Lactobacillus reuteri* XS82-5. The *Lactobacillus reuteri* XS82-5 has the accession number CGMCC No. 39597 and is deposited at the China General Microbiological Culture Collection Center on July 6, 2026. The medicinal and edible composition includes citron, perilla, Ampelopsis japonica leaves, Sophora japonica buds, and double-petaled red rose.

4. The fermentation product according to claim 3, characterized in that, By weight, the medicinal and edible composition includes 30-60 parts of citron, 25-50 parts of perilla, 10-20 parts of Ampelopsis japonica leaf, 1-4 parts of Sophora japonica flower bud, and 10-25 parts of double-petaled red rose.

5. The fermentation product according to claim 3, characterized in that, The enzyme preparations used for enzymatic hydrolysis include cellulase, pectinase, and neutral protease. By weight, it includes 5-10 parts cellulase, 3-8 parts pectinase and 0.5-2 parts neutral protease.

6. The fermentation product according to claim 3, characterized in that, The inoculation amount of *Lactobacillus reuteri* XS82-5 is 5-10%. The viable count of *Lactobacillus reuteri* XS82-5 was 1.0 × 10⁻⁶. 11 CFU / mL or higher.

7. A method for preparing the fermentation product according to any one of claims 3-6, characterized in that, include: (1) The citron, sophora japonica and ampelopsis japonica leaves in the food-medicine homology composition were extracted with water at high temperature to obtain an aqueous extract; (2) Add double-petaled red rose and perilla to the aqueous extract, mix well, add enzyme preparation for enzymatic hydrolysis, and obtain enzymatic hydrolysate; (3) The enzyme hydrolysate is heated to inactivate the enzyme and extract it to obtain an enzyme-inactivated extract; (4) Inoculate the enzyme-inactivating extract with Lactobacillus reuteri XS82-5 for fermentation to obtain the fermentation product; The Lactobacillus reuteri XS82-5 has the accession number CGMCC No.39597 and is deposited at the China General Microbiological Culture Collection Center on July 6, 2026.

8. The method for preparing the fermentation product according to claim 7, characterized in that, The ratio of the medicinal and edible homology composition to water is 1:(10-25).

9. The method for preparing the fermentation product according to claim 7, characterized in that, In step (1), the high-temperature water extraction temperature is 80-100℃ and the water extraction time is 10-20min; In step (2), the enzymatic hydrolysis time is 60-150 min and the enzymatic hydrolysis temperature is 45-60℃; The sterilization temperature in step (3) is 100-120℃ and the time is 20-40 min; The fermentation temperature in step (4) is 25-40℃, and the fermentation time is 30-50h.

10. The use of the fermentation product according to any one of claims 3-6 or the fermentation product prepared by the preparation method according to any one of claims 7-9 in the preparation of products for improving liver qi stagnation.

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