Chinese wolfberry fruit compound fermentation liquor as well as preparation method and application thereof

Abstract

The invention relates to fructus lycii compound fermentation liquor as well as a preparation method and application thereof, and belongs to the technical field of fermentation. According to the preparation method, fructus lycii, rhizoma coptidis, radix notoginseng, radix astragali seu hedysari, rhizoma bletillae, fructus chebulae and rhizoma dioscoreae are mixed and subjected to high-temperature extraction, ackermania muciniphila ATCC BAA-835 is utilized for primary fermentation, lactobacillus paracasei is utilized for secondary fermentation, and the obtained compound fermentation liquor has the advantages that the effect of improving the anti-inflammatory effect is achieved in a complex DSS + bound stress compound type inflammatory bowel disease injury model; according to the invention, all-around protection effect is shown, the efficacy of the lactobacillus plantarum is obviously better than that of unfermented raw materials, any single strain fermented product and a contrast product fermented by adopting strains reported in literature in the same proportion, and important technical support and product prototype are provided for developing functional food for inflammatory bowel diseases and related neuropsychiatric diseases.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a compound fermentation liquid of wolfberry, its preparation method and application. Background Technology

[0002] With the fast pace of modern life, gut health issues are receiving increasing attention. Studies show that gut health problems are becoming increasingly prominent, with complex and diverse causes. Gut microbiota imbalance is closely related to the occurrence of various chronic diseases, such as inflammatory bowel disease (IBD) and metabolic syndrome. Chemical damage, such as dextran sulfate sodium (DSS)-induced colitis, is a classic model for studying IBD; while physiological stress, such as chronic mental stress, activates the hypothalamus-pituitary-adrenal (HPA) axis, disrupting the intestinal barrier and exacerbating gut-microbiota-brain axis dysbiosis. In modern life, these two factors often work synergistically, worsening gut diseases, but existing interventions mostly target single causes, with limited effectiveness in repairing synergistic damage. In recent years, lactic acid bacteria have become a popular area for functional food development due to their excellent probiotic properties and role in regulating the gut microbiota.

[0003] Goji berries (Lycium barbarum), a traditional food and medicine ingredient, have received considerable attention in the field of gut health in recent years. Their core active ingredient—Lycium barbarum polysaccharides (LBPs)—has been proven to have multiple protective effects on the gut: 1) regulating immune balance and inhibiting excessive inflammatory responses; 2) possessing strong antioxidant capabilities, clearing intestinal oxidative stress products; 3) promoting the growth of beneficial bacteria (such as Bifidobacteria and Lactobacillus) and inhibiting opportunistic pathogens; 4) enhancing the intestinal mucosal barrier function and upregulating the expression of tight junction proteins. However, the direct bioavailability of large molecules such as Lycium barbarum polysaccharides is limited, and goji berry puree has a monotonous taste.

[0004] Microbial fermentation technology provides an ideal pathway to integrate and enhance the combined efficacy of the aforementioned medicinal materials. Akkermansia muciniphila (AKK bacteria), an important mucus-degrading bacterium in the gut, produces short-chain fatty acids (SCFAs, especially propionic acid), whose metabolites are crucial for maintaining intestinal barrier integrity and anti-inflammation. However, AKK bacteria require demanding culture conditions and are difficult to apply directly to complex traditional Chinese medicine matrices. Therefore, new fermentation methods are urgently needed. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the direct bioavailability of wolfberry fermentation liquid is limited and the taste of wolfberry pulp is monotonous in the prior art.

[0006] To address the aforementioned technical problems, this invention provides a compound fermentation broth of wolfberry, its preparation method, and its applications. This invention mixes wolfberry, coptis chinensis, notoginseng, astragalus membranaceus, bletilla striata, chebula, and yam, and extracts them at high temperature. Primary fermentation is performed using *Ackermania mutans* ATCC BAA-835, followed by secondary fermentation using *Lactobacillus paracasei*. The resulting compound fermentation broth exhibits comprehensive protective effects in a complex "DSS + restraint stress" intestinal injury model, demonstrating significantly superior efficacy compared to unfermented raw materials, any single-strain fermentation product, and control products fermented with the same proportions of strains reported in the literature. This fully demonstrates the innovation and scientific rigor of this invention in terms of formulation design, process optimization, and strain selection, providing crucial technical support and product prototypes for developing functional foods targeting inflammatory bowel disease and related neuropsychiatric comorbidities.

[0007] The first objective of this invention is to provide a method for preparing a compound fermentation broth, the method comprising the following steps:

[0008] S1. Mix wolfberry, coptis, notoginseng, astragalus, bletilla, chebula and yam and extract at high temperature to obtain the extract;

[0009] S2. Inoculate Akkermansia ATCC BAA-835 into fermentation medium and ferment to obtain fermentation broth. Mix the extract from S1 with the fermentation broth for primary fermentation to obtain primary fermentation product.

[0010] S3. Inoculate Lactobacillus paracasei into the primary fermentation product for secondary fermentation to obtain the composite fermentation broth, wherein the Lactobacillus paracasei includes Lactobacillus paracasei E10, Lactobacillus paracasei JN-1 and Lactobacillus paracasei JN-8.

[0011] Furthermore, the viable cell count in the fermentation broth in step S2 is greater than or equal to 8.6 × 10⁻⁶. 8 CFU / mL.

[0012] Furthermore, the mass ratio of the wolfberry, coptis, notoginseng, astragalus, bletilla, chebula and yam is (15-30): (8-18): (6-15): (12-25): (3-10): (3-6): (5-15).

[0013] Furthermore, the volume ratio of Lactobacillus paracasei E10, Lactobacillus paracasei JN-1 and Lactobacillus paracasei JN-8 is (1-3):(1-3):(1-3).

[0014] Further, in step S1, the high-temperature extraction involves mixing wolfberry, coptis, notoginseng, astragalus, bletilla, chebula and yam, then adding pure water and heating at 90-100℃ for extraction, filtering to obtain a primary filtrate and a primary residue, adding pure water to the primary residue and heating at 90-100℃ for extraction, filtering to obtain a secondary filtrate, mixing the primary filtrate and the secondary filtrate and concentrating to obtain the extract.

[0015] The second objective of this invention is to provide a composite fermentation broth prepared by the above-described preparation method.

[0016] A third objective of this invention is to provide an application of the above-mentioned compound fermentation broth in the preparation of products for the treatment of inflammatory bowel disease.

[0017] Furthermore, the inflammatory bowel disease includes stress-induced inflammatory bowel disease and / or sodium dextran sulfate-induced inflammatory bowel disease.

[0018] The fourth objective of this invention is to provide an application of the above-mentioned composite fermentation broth in the preparation of antioxidant products.

[0019] The fifth objective of this invention is to provide an application of the above-mentioned composite fermentation broth in the preparation of enzyme preparations rich in β-glucosidase.

[0020] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0021] This invention, through optimized design across the entire chain—from the scientific formulation of traditional Chinese medicine compound to the standardized pretreatment of AKK bacteria, and then to the synergistic fermentation of a specific combination of lactic acid bacteria—successfully prepared a dual-strain sequential fermentation beverage with multi-target and multi-level protective effects. In a complex "DSS + restraint stress" complex intestinal injury model, this product demonstrated comprehensive protective effects, with efficacy significantly superior to unfermented raw materials, any single-strain fermentation product, and control products fermented using the same proportions of strains reported in the literature. This fully demonstrates the innovation and scientific rigor of this invention in terms of formulation design, process optimization, and strain selection, providing crucial technical support and a product prototype for the development of functional foods targeting inflammatory bowel disease and related neuropsychiatric comorbidities. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0023] The information on the bacterial strains involved in the following examples is as follows:

[0024] Akkermansia muciniphila ATCC BAA-835 (accession number ATCC BAA-835) is deposited at ATCC (American Type Culture Collection).

[0025] Lactobacillus paracasei E10 is disclosed in patent application number 202310209602.7, accession number CGMCC No.22744.

[0026] Lactobacillus paracasei JN-1 is disclosed in patent application number 202111386682.0, with accession number CGMCC No.22745.

[0027] Lactobacillus paracasei JN-8 is disclosed in patent application number 202210873151.2, accession number CGMCCNo.22746.

[0028] Example 1: Optimization of AKK strain pre-fermentation conditions

[0029] This embodiment first optimizes the fermentation conditions of AKK bacteria on a standardized culture medium to establish a stable and efficient pre-fermentation process, laying a technical foundation for the subsequent pretreatment of traditional Chinese medicine compound extracts.

[0030] 1. Culture medium and bacterial strain:

[0031] A modified BHI medium was used as the basal fermentation substrate, with the following composition: brain and heart extract powder 37.0 g / L, yeast extract 5.0 g / L, peptone 10.0 g / L, glucose 2.5 g / L, L-cysteine ​​hydrochloride 0.5 g / L, heme chloride 10.0 mg / L, vitamin K1 1.0 mg / L, porcine gastric mucin type III 5.0 g / L, pH adjusted to 7.2±0.1, and autoclaved at 121℃ for 15 minutes. The bacterial strain was Akkermansia muciniphila ATCCBAA-835.

[0032] 2. Optimize variables and design:

[0033] To comprehensively investigate the dynamic changes in the growth and metabolites of AKK bacteria, six time gradients were set up: 24h, 36h, 48h, 60h, 72h, and 84h. Three replicates were set up for each time point. The inoculum size was fixed at 5% (v / v), and the cultures were statically incubated in an anaerobic workstation (85% N2, 10% CO2, 5% H2) at 37℃.

[0034] 3. Evaluation Indicators and Results:

[0035] The evaluation was conducted using two indicators: viable bacterial count and the production of the key metabolite propionic acid.

[0036] 4. Conclusions and Process Determination:

[0037] According to the data in Table 1, the viable count of AKK bacteria reached a peak of 8.9 × 10⁻⁶ after 48 hours of fermentation. 8 The CFU / mL concentration yielded a relatively high propionic acid production of 3.8 mmol / L, with the highest specific growth rate. Further extending the fermentation time led to a decrease in viable cell count, while the increase in propionic acid production slowed. Therefore, the AKK standard pre-fermentation process (S-ACK) was determined to be: inoculating 5% AKK bacteria into modified BHI medium (containing 0.5% mucin) and fermenting under anaerobic conditions at 37℃ for 48 hours. The AKK bacteria fermentation broth obtained under these conditions (rich in highly active cells and propionic acid) can be used as a highly efficient pretreatment agent for subsequent traditional Chinese medicine compound extracts.

[0038] Table 1: Effects of different fermentation times on the growth and metabolism of AKK bacteria in standard culture medium

[0039]

[0040] Note: Data are expressed as mean ± standard deviation (n=3). 1) This indicates that compared with the 24h, 36h, 60h, 72h, and 84h groups, P<0.05.

[0041] Example 2: Determination of the ratio of compound raw materials

[0042] Based on the established standard fermentation process of AKK bacteria, we conduct scientific formulation and standardized extraction of traditional Chinese medicine compound prescriptions.

[0043] Based on traditional Chinese medicine theory and modern pharmacology, this embodiment designed a traditional Chinese medicine compound with wolfberry as the core, in combination with Coptis chinensis, Panax notoginseng, Astragalus membranaceus, Bletilla striata, Terminalia chebula, and Dioscorea opposita. Through five parallel experiments with different ratios (G1-G5), the optimal compound composition was determined using in vitro comprehensive antioxidant capacity and anti-inflammatory activity as dual screening indicators.

[0044] Standardized extraction and AKK pretreatment:

[0045] Extraction: Weigh the mixed medicinal materials according to the proportions in Table 2, add 10 times the amount (w / v) of pure water, reflux at 95℃ for 1.5 hours, and filter. Add 8 times the amount of water to the residue, extract again in the same way for 1 hour, and filter. Combine the filtrates, concentrate under reduced pressure at 60℃ to a crude drug concentration of 80 g / L, and sterilize at 115℃ for 20 minutes to obtain standardized extracts (labeled as Ex-G1 to Ex-G5).

[0046] Table 2 Screening of compound Chinese medicine formulas containing wolfberry

[0047]

[0048] AKK pretreatment: Take 3 volumes of extracts from Ex-G1 to Ex-G5 respectively, and add 1 volume of AKK fermentation supernatant prepared by the S-ACK process (viable cell count approximately 8.6 × 10⁻⁶). 8 The mixture (CFU / mL) was thoroughly mixed and incubated at 37°C for 24 hours to obtain the pretreated reaction mixture (labeled as PM-G1 to PM-G5).

[0049] Evaluation indicators and methods:

[0050] DPPH free radical scavenging rate: reflects the ability to directly scavenge free radicals.

[0051] ABTS⁺·Cation radical scavenging ability: Reflects the overall antioxidant capacity of hydrophilic and lipophilic components.

[0052] Ferrous ion reducing power (FRAP): reflects the reduction potential of a sample.

[0053] Anti-inflammatory activity: The inhibition rate of nitric oxide (NO) production was measured using an LPS-induced RAW264.7 macrophage model.

[0054] Four indicators of each group of pretreatment reaction mixtures (PM-G1 to PM-G5) were measured, and a comprehensive score was calculated.

[0055] The results are shown in Table 3. The G3 formulation (20g Lycium barbarum, 15g Coptis chinensis, 12g Panax notoginseng, 20g Astragalus membranaceus, 8g Bletilla striata, 5g Terminalia chebula, and 12g Dioscorea opposita) was significantly superior to other formulations in all antioxidant indicators, anti-inflammatory activity, and overall score (P < 0.05). This indicates that this formulation has the best potential in providing the basis for antioxidant and anti-inflammatory substances. Therefore, the G3 formulation was determined to be the optimal compound (OF-GLB, Lycium barbarum-based optimized compound) for subsequent core fermentation.

[0056] Table 3 Comparison of antioxidant activities of water extracts with different raw material ratios (G1-G5)

[0057]

[0058] *Note: Data are expressed as mean ± standard deviation (n=3). 1) This indicates that compared with groups G1, G2, G4, and G5, P < 0.05. The overall score was normalized, with group G3 set to 1.00.

[0059] Example 3: Lactic acid bacteria compound fermentation based on optimized intermediate product M-OPT

[0060] PM-G3, obtained by pretreating OF-GLB (G3 ratio) extract with the above-mentioned AKK, was used as the unified substrate for the primary fermentation (labeled as M-GOPT) for the second fermentation, aiming to further enhance the product activity by utilizing the lactic acid bacteria combination (E10, JN-1 and JN-8).

[0061] 1. Experimental design for optimizing the ratio of lactic acid bacteria:

[0062] With a fixed total inoculum of 5% (v / v), and using M-GOPT as the sole fermentation substrate, five groups of lactic acid bacteria volume ratios were designed for comparative fermentation (37℃, 48h):

[0063] Group LP1: E10 : JN-1 : JN-8 = 2 : 1 : 1

[0064] Group LP2: E10 : JN-1 : JN-8 = 3 : 2 : 1

[0065] Group LP3: E10 : JN-1 : JN-8 = 3 : 3 : 2

[0066] Group LP4: E10 : JN-1 : JN-8 = 4 : 3 : 2

[0067] LP5 group (equal proportion control): E10 : JN-1 : JN-8 = 1 : 1 : 1

[0068] Group LP6: L. paracasei CCFM1222, L. paracasei CCFM1223, and L. casei YRL577, fermented in a 3:3:2 ratio. (L. paracasei CCFM1222 is disclosed in the paper "Lactobacillus paracaseiCCFM1222 Ameliorated the Intestinal Barrier and Regulated Gut Microbiota in Mice with Dextran Sulfate Sodium-Induced Colitis", L. paracasei CCFM1223 is disclosed in patent literature (CN114854638A), and L. casei YRL577 is disclosed in the paper "Lactobacillus casei YRL577 ameliorates markers of non-alcoholic fatty liver and alters expression of genes within the intestinal bile acid pathway").

[0069] 3.2 Evaluation Indicators:

[0070] After fermentation, the following key indicators of the fermentation broth in each group were measured and compared:

[0071] Fermentation performance: endpoint pH, total number of viable lactic acid bacteria.

[0072] Enzyme activity: β-glucosidase activity (reflects the ability to convert flavonoid glycosides and other components).

[0073] Changes in antioxidant activity: Percentage increase in DPPH radical scavenging rate compared to M-GOPT. Changes in total phenolic content: Percentage increase in total phenolic content (calculated as gallic acid, GAE) compared to M-GOPT.

[0074] 3.3 Conclusion:

[0075] Based on the results in Table 4, the superiority of the strain combination (LP3) of this invention is as follows: LP3 group (E10:JN-1:JN-8 = 3:3:2) showed the best and most significant performance in all evaluation indicators. Its fermentation endpoint pH was the lowest (4.0), indicating strong acid production capacity; and its viable lactic acid bacteria count was the highest (5.9 × 10⁻⁶). 9The CFU / mL count indicated vigorous growth; its β-glucosidase activity, antioxidant capacity (DPPH scavenging rate increased by 33.7%), and total phenol content (increased by 27.1%) were significantly higher than all other ratio groups, including the LP6 strain combination reported in the literature (P < 0.05).

[0076] Table 4 Comparison of fermentation effects of different lactic acid bacteria ratios (LP1-LP5) based on M-OPT

[0077]

[0078] Note: 1) The results showed that all indicators of group R3 (E10:JN-1:JN-8=3:3:2) were significantly better than those of all other ratio groups (R1, R2, R4, R5, R6, R7, R8, R9), P < 0.05.

[0079] The LP6 group used the exact same ratio as the LP3 group (3:3:2), but the strains were replaced with *L. paracasei* CCFM1222, *L. paracasei* CCFM1223, and *L. casei* YRL577, which have been reported to have probiotic effects. The experimental results showed that all fermentation performance indicators (viable cell count, enzyme activity) and functional indicators (antioxidant activity, increased total phenols) of the LP6 group were significantly lower than those of the LP3 group, and there was no significant difference or only slightly lower values ​​compared to the LP5 group (equal proportion E10 combination).

[0080] Example 4: Preparation of AKK bacteria standard pre-fermentation broth

[0081] Step 1: Culture medium preparation

[0082] The modified BHI medium consisted of the following: brain and heart extract powder 37.0 g / L, yeast extract 5.0 g / L, peptone 10.0 g / L, glucose 2.5 g / L, L-cysteine ​​hydrochloride 0.5 g / L, heme chloride 10.0 mg / L, vitamin K1 1.0 mg / L, and porcine gastric mucin type III 5.0 g / L. The medium was brought to a final volume of 1 L with deionized water, the pH was adjusted to 7.2 ± 0.1, and the medium was autoclaved at 121°C for 15 minutes. It was then cooled and stored for later use.

[0083] Step 2: Activation of microbial strains

[0084] Take the frozen Akkermansia muciniphila ATCC BAA-835 strain, inoculate it into the above culture medium, and incubate it statically in an anaerobic workstation at 37℃ (85% N2, 10% CO2, 5% H2) for 48 hours, and continuously activate it for two generations.

[0085] Step 3: Pre-fermentation

[0086] The activated bacterial solution was inoculated into fresh culture medium at a 5% (v / v) inoculation rate and incubated statically for 48 hours under the same anaerobic conditions. After fermentation, the viable cell count reached 8.9 × 10⁻⁶. 8 The AKK standard pre-fermentation broth (S-ACK) was obtained with CFU / mL, a propionic acid yield of 3.8 mmol / L, and a pH of 6.2±0.1, and was ready for use.

[0087] Example 5: Preparation of compound extracts from traditional Chinese medicine and AKK pretreatment

[0088] Step 1: Extract Preparation

[0089] Weigh out 20 g of wolfberry, 15 g of coptis, 12 g of notoginseng, 20 g of astragalus, 8 g of bletilla, 5 g of chebula, and 12 g of yam (total 92 g) according to the G3 ratio in Table 1. Add 920 mL of pure water (10 times the volume) and extract by reflux at 95℃ for 1.5 hours, then filter. Add 736 mL of water (8 times the volume) to the residue and extract again by the same method for 1 hour, then filter. Combine the two filtrates and concentrate under reduced pressure at 60℃ to a total volume of approximately 1150 mL (crude drug concentration 80 g / L). Sterilize at 115℃ for 20 minutes to obtain the standardized extract.

[0090] Step 2: AKK Preprocessing

[0091] Take 300 mL of Ex-G3 extract and add 100 mL of S-ACK fermentation broth prepared in Example 4 (with a viable count of approximately 8.6 × 10⁻⁶). 8 The mixture (CFU / mL) was thoroughly mixed and incubated at 37°C for 24 hours to obtain the pretreated reaction mixture (labeled PM-G3, i.e., M-GOPT matrix).

[0092] Example 6: Lactic acid bacteria compound fermentation process

[0093] Step 1: Preparation of Lactic Acid Bacteria Seed Culture

[0094] Lacticaseibacillus paracasei E10, L. paracasei JN-1, and L. paracasei JN-8 were inoculated into MRS liquid medium and incubated statically at 37°C for 24 hours. After two generations of activation, seed cultures of each strain (viable count ≥1×10⁻⁶) were obtained. 9 (CFU / mL).

[0095] Step 2: Single-strain and compound fermentation

[0096] 500 mL of the PM-G3 substrate prepared in Example 5 was inoculated into a mixed seed culture of lactic acid bacteria at a 5% inoculation rate, wherein the volume ratio of E10, JN-1, and JN-8 was 3:3:2. The culture was then incubated at 37°C for 48 hours. At the end of fermentation, the final pH was measured to be 4.0 ± 0.1, and the viable count of lactic acid bacteria reached 5.9 × 10⁻⁶. 9 The concentration of CFU / mL was increased, the β-glucosidase activity was 1.28 U / mL, the DPPH scavenging rate was increased by 33.7% compared with that before fermentation, and the total phenol content was increased by 27.1%.

[0097] Step 3: Post-processing

[0098] The fermentation broth was centrifuged at 5000 rpm for 10 minutes to remove bacteria. The supernatant was filtered through a 0.22 μm filter membrane for sterilization to obtain a clarified fermentation broth, which is the original liquid of the fermented beverage with enteritis protection function described in this invention. After being dispensed and sterilized, the final product (marked as DF-GOPT) is obtained.

[0099] 3. Mental stress and DSS-induced inflammatory bowel disease mouse experiment: This experiment used specific pathogen-free (SPF) grade female Balb / c mice, 6 weeks old, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (License No.: SCXK (Su) 2023-0009). The mice were housed in an SPF grade experimental barrier environment at the Experimental Animal Center of Jiangnan University, with the temperature maintained at 22 ± 2 ℃ and the relative humidity at 55 ± 10%, and a 12-hour light-dark cycle was implemented. The animal use and care protocols involved in the experiment complied with the EU Laboratory Animal Guidelines (Directive 2010 / 63 / EU) and were approved by the Animal Welfare and Ethics Committee of Jiangnan University (Ethics Approval No.: JN.No20230615b0800801

[258] ).

[0100] The study on stress and DSS-induced inflammatory bowel disease in mice included 9 experimental groups, with 5 mice in each group:

[0101] (1) Control group (CTL): Adaptive feeding for 6 days, followed by free access to sterile purified water, and daily gavage with physiological saline at a dose of 5 mL / kg.

[0102] (2) DSS-induced inflammatory bowel disease model group (DSS): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS for 7 consecutive days, and physiological saline was administered by gavage at a dose of 5 mL / kg per day.

[0103] (3) Chronic restraint model group (CRS): After 6 days of adaptive feeding, the restraint stress method was used daily from day 1 to day 7. Mice were restrained for 4 hours daily in a well-ventilated self-made behavioral restraint device to simulate mental stress. Physiological saline was administered by gavage at a dose of 5 mL / kg daily.

[0104] (4) Chronic restraint + DSS-induced inflammatory bowel disease model group (DSS+CRS): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS. The restraint stress method was used daily, and the mice were placed in a well-ventilated self-made behavioral restraint device for 4 hours daily to simulate mental stress, and physiological saline was administered by gavage at a dose of 5 mL / kg.

[0105] (5) Unfermented compound water extract intervention group (DSS+CRS+OF-GLB): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS. The restraint stress method was used daily, and the mice were restrained in a well-ventilated self-made behavioral restraint device for 4 hours daily to simulate mental stress. Unfermented compound water extract was administered by gavage at a dose of 5 mL / kg.

[0106] (6) E10 single-strain fermented beverage intervention group (DSS+CRS+Akk-F): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS. The restraint stress method was used daily, and the mice were restrained in a well-ventilated self-made behavioral restraint device for 4 hours daily to simulate mental stress. Akk single-strain pre-fermented OF-GLB extract was administered by gavage at a dose of 5 mL / kg.

[0107] (7) E10 single-strain fermented beverage intervention group (DSS+CRS+E10-F): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS. The restraint stress method was used daily, and the mice were restrained in a well-ventilated self-made behavioral restraint device for 4 hours daily to simulate mental stress. The mice were also given E10 single-strain fermented M-GOPT substrate by gavage at a dose of 5 mL / kg.

[0108] (8) JN-1 single-strain fermented beverage intervention group (DSS+CRS+JN-1): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS. The restraint stress method was used daily, and the mice were restrained in a well-ventilated self-made behavioral restraint device for 4 hours daily to simulate mental stress. JN-1 single-strain fermented M-GOPT substrate was administered by gavage at a dose of 5 mL / kg.

[0109] (9) JN-8 single-strain fermented beverage intervention group (DSS+CRS+JN-8-F): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS. The restraint stress method was used daily, and the mice were restrained in a well-ventilated self-made behavioral restraint device for 4 hours daily to simulate mental stress. JN-8 single-strain fermented M-GOPT substrate was administered by gavage at a dose of 5 mL / kg.

[0110] (10) Compound fermented beverage intervention group (DSS+CRS+LP3-F): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS. The restraint stress method was used daily, and the mice were restrained in a well-ventilated self-made behavioral restraint device for 4 hours daily to simulate mental stress. The compound fermented wolfberry pulp beverage prepared by the mixed strain of the present invention according to LB3 was administered by gavage at a dose of 5 mL / kg.

[0111] (11) Compound fermented beverage intervention group (DSS+CRS+LP6-F): Adaptive feeding for 6 days, followed by free access to sterile purified water containing 2.5% (w / v) DSS. The restraint stress method was used daily, and the mice were restrained in a well-ventilated self-made behavioral restraint device for 4 hours daily to simulate mental stress. The mice were also given a compound fermented wolfberry pulp beverage prepared by the reported strain according to the LP6 ratio by gavage at a dose of 5 mL / kg.

[0112] On day 8, after all mice were fasted for 12 hours but allowed free access to water, they were euthanized by isoflurane anesthesia, and their eyeballs were removed for blood collection for subsequent serological studies. Their colons were also dissected for analysis.

[0113] Indicator Testing:

[0114] Neurological indicators: Measurement of anxiety behavior in mice (forced swimming test, tail suspension test);

[0115] Oxidative stress indicators: measuring the levels of serotonin (5-HT), corticosterone (CORT), and adrenaline (EPI) in the brain (hippocampus).

[0116] Indicators of colonic injury: The relative mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-6) and mucin (Muc2) in colonic tissue were measured.

[0117] Experimental results:

[0118] This animal experiment fully demonstrates that:

[0119] 1. Model Validity

[0120] A single damage model was successfully established:

[0121] DSS-induced colitis model: Mice showed significant colonic shortening (-34.2%), increased DAI score (3.20±0.21), upregulated expression of pro-inflammatory factors TNF-α and IL-6 by 482% and 376%, respectively, and downregulated expression of intestinal barrier proteins ZO-1 and MUC2 by 68% and 75%, respectively. They also exhibited depressive-like behavior (prolonged forced swimming time by 71.1%) and neurochemical disorders (decreased 5-HT levels by 48.2% and increased CORT levels by 87.5%).

[0122] The chronic restraint stress model mainly resulted in neurobehavioral abnormalities (forced swimming time increased by 47.4%, tail suspension time increased by 51.1%) and neuroendocrine disorders (5-HT decreased by 33.0%, CORT increased by 54.8%), with relatively mild intestinal damage.

[0123] Synergistic aggravation effect of complex damage model:

[0124] The DSS+CRS combined group showed more severe impairment in all indicators than the single model (P < 0.05), particularly: a further 5.9% reduction in colon length, a 20.3% increase in DAI score, a 19.4% increase in TNF-α expression, a 27.1% prolongation in forced swimming time, a further 15.9% decrease in 5-HT levels, and a 33.2% increase in CORT levels.

[0125] This composite model successfully simulated the synergistic pathological state of "intestinal inflammation + mental stress", providing a more realistic disease model for the comprehensive evaluation of the product of this invention.

[0126] Table 5. Changes in body weight of mice in each group during the DSS-induced colitis model experiment.

[0127]

[0128] Note: Compared with the normal group 1) P<0.05

[0129] 2. Gradient analysis of intervention effect

[0130] (a) Intestinal protective effect

[0131] All intervention groups showed varying degrees of improvement in the combined DSS+CRS damage, but the effects exhibited a clear gradient:

[0132] Unfermented raw material group (DSS+CRS+OF-GLB): Colon length recovery rate: 14.0% (vs. DSS+CRS model group); DAI score reduction: 28.6%; Inhibition of pro-inflammatory factors: TNF-α reduced by 30.2%, IL-6 reduced by 31.3%; Barrier protein recovery: ZO-1 increased by 92.0%, MUC2 increased by 125.0%.

[0133] The AKK bacteria pretreatment group (DSS+CRS+Akk-F) showed better results than the raw material group, with a colon length recovery rate increasing to 22.1%, indicating that AKK bacteria pretreatment can effectively modify the substrate of traditional Chinese medicine and enhance its biological activity.

[0134] Single-strain fermentation groups (DSS+CRS+E10-F / JN-1-F / JN-8-F): Further improvements were observed, with the E10 single-strain group showing the best performance: colon length recovery rate: 24.6%; DAI score reduction: 44.9%; TNF-α reduction: 44.6%; ZO-1 recovery: 160.0%.

[0135] The literature strain combination group (DSS+CRS+LP6-F) showed comparable results to the best single strain group (E10-F), with no significant synergistic advantage, indicating that simply referencing the literature strain combination cannot achieve results superior to single strains.

[0136] The experimental group (DSS+CRS+LP3-F) showed the best and significantly better results than all other intervention groups in all intestinal indicators: colon length recovery rate: 39.2%, close to the normal group level; DAI score reduction: 67.5%, significantly improving clinical symptoms. Inhibition of pro-inflammatory factors: TNF-α reduced by 66.2%, IL-6 reduced by 66.7%. Restoration of barrier proteins: ZO-1 recovered to 88% of the normal group, MUC2 recovered to 90%.

[0137] Table 6. Comparison of colon length and disease activity index (DAI) of mice in each group at the experimental endpoint (day 8).

[0138]

[0139] Note: Compared with the normal group 1) P<0.05; compared with the normal group 2) P<0.01; compared with the model group (DSS+CRS) 3) P<0.05; compared with the model group (DSS+CRS) 4) P<0.01

[0140] Table 7. Relative mRNA expression levels of inflammatory factors, tight junction proteins, and mucins in colon tissue detected by qRT-PCR (CTL group = 1.00, mean ± SD, n = 5).

[0141]

[0142] Note: Compared with the normal group 1) P<0.05; compared with the normal group 2) P<0.01; compared with the model group (DSS+CRS)3) P<0.05; compared with the model group (DSS+CRS) 4) P<0.01

[0143] 3. Neurobehavioral improvement effects

[0144] The composite fermentation broth in this embodiment also demonstrated remarkable effects in improving neurobehavioral abnormalities related to the gut-brain axis: Behavioral improvements: Forced swimming test: immobility time was reduced by 43.0%, an improvement 1.2 times that of the optimal single-strain group (E10-F). Tail suspension test: immobility time was reduced by 46.0%, an improvement 1.2 times that of the optimal single-strain group. Neurochemical regulation: 5-hydroxytryptamine (5-HT) level: recovered to 93.0% of the normal group, an increase of 114% (vs. model group). Corticosterone (CORT) level: decreased to 128.4% of the normal group, a decrease of 48.6%. The regulatory effect was significantly better than that of each single-strain group and the literature bacterial combination group (P < 0.05).

[0145] Table 8 Comparison of behavioral indicators of mice in each group (mean ± SD, n=5)

[0146]

[0147] Note: Compared with the normal group 1) P<0.05; compared with the normal group 2) P<0.01; compared with the model group (DSS+CRS) 3) P<0.05; compared with the model group (DSS+CRS) 4) P<0.01.

[0148] Table 9 Comparison of neurochemical indices in the hippocampus of mice in each group (mean ± SD, n=5)

[0149]

[0150] Note: Compared with the normal group 1) P<0.05; compared with the normal group 2) P<0.01; compared with the model group (DSS+CRS) 3) P<0.05; compared with the model group (DSS+CRS) 4) P<0.01

[0151] 4. Key control verification

[0152] Specificity verification of the strain combination: Under the same fermentation ratio (3:3:2), the strain combination of the present invention (E10 / JN-1 / JN-8) is significantly better than the strain combination reported in the literature (CCFM1222 / CCFM1223 / YRL577). In terms of key indicators, the combination of the present invention is 35-45% better than the combination in the literature, which proves the unique synergistic metabolic ability and irreplaceability of the strain combination of the present invention in fermenting specific Chinese medicine matrices.

[0153] Comparative Example 1: Unfermented Compound Aqueous Extract

[0154] Only the compound Chinese medicine from Example 2 was subjected to water extraction.

[0155] Comparative Example 2: OF-GLB extract pre-fermented by Akk single strain

[0156] Only Akk single-strain pre-fermentation as described in Example 5 was performed; secondary lactic acid bacteria fermentation was not performed.

[0157] Comparative Example 3: E10 single-strain fermentation of M-GOPT substrate

[0158] Replace the lactic acid bacteria mixed seed liquid in Example 6 with E10, and keep the other steps unchanged.

[0159] Comparative Example 4: JN-1 single-strain fermentation of M-GOPT substrate

[0160] Replace the lactic acid bacteria mixed seed liquid in Example 6 with JN-1, and keep the other steps unchanged.

[0161] Comparative Example 5: JN-8 single-strain fermentation of M-GOPT substrate

[0162] Replace the lactic acid bacteria mixed seed liquid in Example 6 with JN-8, and keep the other steps unchanged.

[0163] Comparative Example 6: A compound fermented wolfberry pulp beverage prepared at a ratio of LP6

[0164] Replace the lactic acid bacteria mixed seed liquid of Example 6 with the bacterial strain of group LP6, and keep the other steps unchanged.

[0165] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a compound fermentation broth, characterized in that, The preparation method includes the following steps: S1. Mix wolfberry, coptis, notoginseng, astragalus, bletilla, chebula and yam and extract at high temperature to obtain the extract; S2. Inoculate Akkermansia ATCC BAA-835 into fermentation medium and ferment to obtain fermentation broth. Mix the extract from S1 with the fermentation broth for primary fermentation to obtain primary fermentation product. S3. Inoculate Lactobacillus paracasei into the primary fermentation product for secondary fermentation to obtain the composite fermentation broth, wherein the Lactobacillus paracasei includes Lactobacillus paracasei E10, Lactobacillus paracasei JN-1 and Lactobacillus paracasei JN-8.

2. The preparation method according to claim 1, characterized in that, The viable cell count in the fermentation broth in step S2 is greater than or equal to 8.6 × 10⁻⁶. 8 CFU / mL.

3. The preparation method according to claim 1, characterized in that, The mass ratio of the following ingredients is (15-30): (8-18): (6-15): (12-25): (3-10): (3-6): (5-15).

4. The preparation method according to claim 1, characterized in that, The volume ratio of Lactobacillus paracasei E10, Lactobacillus paracasei JN-1 and Lactobacillus paracasei JN-8 is (1-3):(1-3):(1-3).

5. The preparation method according to claim 1, characterized in that, In step S1, the high-temperature extraction involves mixing wolfberry, coptis, notoginseng, astragalus, bletilla, chebula and yam, then adding pure water and heating at 90-100℃ for extraction. The mixture is filtered to obtain a primary filtrate and a primary residue. Pure water is added to the primary residue and heated at 90-100℃ for extraction. The mixture is then filtered to obtain a secondary filtrate. The primary and secondary filtrates are mixed and concentrated to obtain the extract.

6. The composite fermentation broth prepared by the preparation method according to any one of claims 1-5.

7. The use of the compound fermentation broth according to claim 6 in the preparation of products for the treatment of inflammatory bowel disease.

8. The application according to claim 7, characterized in that, The inflammatory bowel disease includes stress-induced inflammatory bowel disease and / or sodium dextran sulfate-induced inflammatory bowel disease.

9. The application of the composite fermentation broth according to claim 6 in the preparation of antioxidant products.

10. The use of the composite fermentation broth according to claim 6 in the preparation of enzyme preparations rich in β-glucosidase.

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