Fermented lactobacillus muci, composition and application

Abstract

The application discloses fermented lactobacillus muciiparus, a composition and application. The fermented lactobacillus muciiparus is classified as lactobacillus muciiparus Limosilactobacillus fermentum , with strain number ZD-011 and preservation number CCTCC NO: M 20251136. The strain has double activities of promoting theanine absorption and self-antidepressant. The fermented lactobacillus muciiparus ZD-011 has a synergistic action mode with the theanine, and the antidepressant effect produced by the combination of the two is significantly better than the sum of the effects of the two when used alone, thereby providing a new and more advantageous combination scheme for developing efficient and composite antidepressant products. The strain and the composition are both derived from nature, have high safety, avoid many side effects of traditional chemical drugs, and can be applied to the fields of auxiliary treatment drugs and the like, and have wide market prospects and social values.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a fermenting Lactobacillus mucinus, its composition, and its applications. Background Technology

[0002] Depression is a common mood disorder with a high global incidence, seriously endangering human physical and mental health. Currently, mainstream treatments (such as SSRIs) often have various side effects, including delayed onset of action, sexual dysfunction, and weight gain, leading to poor patient adherence. Therefore, developing safe and effective alternative or adjunctive therapies is of great significance.

[0003] Theanine is a non-protein amino acid unique to tea. Numerous studies have shown that it has physiological functions such as relieving anxiety, improving sleep, and stabilizing mood, and it is extremely safe. However, the bioavailability of theanine after oral administration is relatively limited, and there are significant individual differences, which affects the stable exertion of its efficacy and the determination of the optimal dosage.

[0004] In recent years, the "gut-brain axis" theory has provided a new perspective for the treatment of mental illnesses. Gut probiotics can improve host mood and behavior through various mechanisms, including regulating the immune system, reducing inflammation levels, producing neuroactive metabolites (such as GABA and short-chain fatty acids), and influencing vagal nerve signal transduction. Furthermore, some probiotic strains have been shown to alter the host's metabolic state, enhancing the digestion, absorption, and utilization of specific nutrients.

[0005] The invention application with publication number CN116814464A discloses a strain of fermenting Lactobacillus mucinus JF5 and its application in the preparation of fat-reducing and digestive aid foods and medicines. This bacterium has probiotic functions such as promoting the digestion and absorption of protein foods to improve protein allergies, promoting the absorption of cellulose to improve constipation, anti-depression and hangover relief, anti-aging and anti-wrinkle, lowering blood sugar, and promoting intestinal absorption and peristalsis.

[0006] The invention application with publication number CN119700832A discloses the application of a fermenting Lactobacillus strain A21196 in the preparation of products that are anti-anxiety and improve memory and concentration. The fermenting Lactobacillus strain A21196 has the effects of anti-anxiety, anti-depression, improving cognitive impairment and improving memory and concentration.

[0007] Chinese patent application CN119876281A discloses the application of *Lactobacillus myxoides* B14.32 in fermented traditional Chinese medicine and an antidepressant fermented composition of traditional Chinese medicine. It combines microbial fermentation technology with traditional Chinese medicine extraction, using *Lactobacillus myxoides* B14.32 and yeast to ferment traditional Chinese medicine with antidepressant effects in a two-step process to obtain an antidepressant fermented composition. This maximizes the preservation of the active ingredients of the traditional Chinese medicine, enhances efficacy, and reduces toxicity. The obtained antidepressant fermented composition has antidepressant effects and reduces brain inflammation in depressed patients. However, current research on the combination of probiotics and theanine is mostly based on the simple superposition of their independent functions, lacking the discovery and development of specific probiotic strains that can produce a clear synergistic effect with theanine and also possess antidepressant activity themselves.

[0008] For example, invention application CN113332318A discloses a pharmaceutical composition and its preparation method that improves depressive symptoms. The composition contains 50-80% of a blanching water extract of enoki mushrooms, 1-3% theanine, 1-10% probiotics, 5-30% prebiotics, 5-10% sugar substitute, and a pharmaceutically acceptable carrier. The probiotics are selected from any one or a combination of Lactobacillus casei, yeast, probiotic spores, Clostridium butyricum, Lactobacillus, Bifidobacterium lactis, Streptococcus thermophilus, and actinomycetes. Summary of the Invention

[0009] This invention obtained a fermenting lactobacillus strain with dual activities of "promoting theanine absorption" and "self-antidepressant".

[0010] This invention first provides a fermenting *Lactobacillus mucinus*, classified and named *Lactobacillus mucinus*. Limosilactobacillus fermentum , Plant No. ZD-011, Preservation No. CCTCC NO: M 20251136.

[0011] This invention further provides the application of the aforementioned fermented *Lactobacillus mucinus* in the preparation of antidepressant products. Preferably, the product is a pharmaceutical or animal feed.

[0012] The present invention also provides a composition comprising theanine and the fermented Lactobacillus mucinus.

[0013] Preferably, in the composition, each 1.2 mg of theanine corresponds to 1 × 10 7 ~1×10 11 The CFU contains live fermented *Lactobacillus mucinus* bacteria. More preferably, each 1.2 mg of theanine corresponds to 2 × 10⁻⁶ bacteria. 8 The CFU contains live fermented *Lactobacillus mucinus* bacteria.

[0014] Preferably, the composition further comprises a pharmaceutically acceptable carrier and / or prebiotic.

[0015] Preferably, the composition is in the form of powder, granules, capsules, tablets, or oral liquid.

[0016] The present invention also provides the use of the composition in the preparation of antidepressant products. Preferably, the product is a pharmaceutical or animal feed.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. A functionally defined fermenting Lactobacillus ZD-011 was obtained, which has dual activities of "promoting theanine absorption" and "self-antidepressant".

[0019] 2. This study reveals for the first time the unique synergistic effect between fermented Lactobacillus mucinus ZD-011 and theanine: the antidepressant effect produced by the combination of the two is significantly better than the sum of the effects of using them alone, providing a new and more advantageous combination scheme for the development of highly effective compound antidepressant products.

[0020] 3. The strains and compositions described herein are all derived from natural sources, have high safety, and avoid many side effects of traditional chemical drugs. They can be applied in fields such as adjuvant therapy drugs and have broad market prospects and social value. Attached Figure Description

[0021] Figure 1 Streak diagram of Lactobacillus fermentum ZD-011.

[0022] Figure 2 Example 2 illustrates the effect of Lactobacillus mucin ZD-011 fermentation on behavioral indicators in a corticosterone-induced depression model mouse. Among these, Figure 2 (A) in the middle: Open field experiment; Figure 2 (B) in the figure: tail suspension experiment. * indicates p < 0.05 compared with the model group, and ** indicates p < 0.01 compared with the model group.

[0023] Figure 3 Example 3: The time-dependent curve of serum theanine concentration in mice after oral administration of theanine (60 mg / kg) is shown.

[0024] Figure 4 Comparison of drug-time curves showing the change of theanine concentration in the serum of mice in the control group (theanine) and the experimental group (theanine + fermented Lactobacillus mucin ZD-011) over time.

[0025] Figure 5 Example 5: A comparative bar chart of corticosterone-induced depression model mice after different interventions. Figure 5 (A) in the middle: Open field experiment; Figure 5 (B) in the middle: Tail suspension test; Figure 5(C): Sucrose preference experiment. # indicates p < 0.05 compared to the normal group, ### indicates p < 0.001 compared to the normal group, #### indicates p < 0.0001 compared to the normal group, * indicates p < 0.05 compared to the model group, ** indicates p < 0.01 compared to the model group, *** indicates p < 0.001 compared to the model group, **** indicates p < 0.0001 compared to the model group. Detailed Implementation

[0026] Example 1: Identification and Preservation of Strains ZD-011

[0027] One sample of feces from a healthy person was collected using a disposable sterile sampler. After serial dilution, the sample was spread onto MRS medium containing 0.05% nystatin and incubated for 48 hours. After incubation, colonies were selected based on color, size, and edge shape using an inoculation loop and streaked for purification. Figure 1 .

[0028] Molecular biological identification: Genomic DNA was extracted from the strain, its 16S rRNA gene sequence was amplified and sequenced. The obtained sequence was BLAST-aligned in the NCBI database. Limosilactobacillus fermentum The homology exceeds 99%. The strain sequence is as follows:

[0029] CGACGTGTGCTATACTGCAAGTCGAACGCGTTGGCCCAATTGATTGATGGTGCTTGCACCTGATTGATTTTGGTTGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAGCGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGGTTCCGCCCTTCAGGGCCGGAGTTAAGCATTAAGCACTCGCCCGGGGAGACCACCGCCAGGGTGAAATCCAAGGAATTTCGGGGGCCCCCCAAAGGGGGGAGATGTG。

[0030] Strain preservation: The pure culture of this strain was submitted to the China Center for Type Culture Collection (CCTCC) for preservation at Wuhan University, Wuhan, China, on May 20, 2025, and classified as *Lactobacillus fermentatus*. Limosilactobacillus fermentum , Plant No. ZD-011, Preservation No. CCTCC NO: M 20251136.

[0031] Example 2: Verification of the antidepressant effect of fermented Lactobacillus mucinus ZD-011

[0032] Establishment of the depression model: A chronic corticosterone-induced depression model was established in C57BL / 6J mice. Corticosterone (CAS: 50-22-6, purity ≥98%) was dissolved in an aqueous solution containing 0.45% (w / v) hydroxypropyl-β-cyclodextrin (HP-β-CD), with a corticosterone concentration of 0.1 mg / mL. The solution was provided to the mice for free access to the water, with a daily intake of approximately 5 mg / kg body weight for 4 weeks.

[0033] Experimental grouping and intervention: Mice were randomly divided into 3 groups, with 6 mice in each group:

[0034] Table 1. Animal Experiment Groups of the Antidepressant Effect of Fermented Lactobacillus mucinus ZD-011

[0035]

[0036] Behavioral testing: conducted 4 weeks after intervention.

[0037] Open field test (OFT): Mice were gently placed in the center of the bottom of a square open field box (40 cm × 40 cm × 40 cm) and allowed to explore freely for 5 minutes. A video tracking system recorded the total distance the mice traveled and the time and distance spent in the central area (defined as the area within 10 cm of the four walls).

[0038] Tail Suspension Test (TST): The tail of a mouse was secured with tape approximately 2 cm from the tip, and the mouse was suspended upside down in a box about 30 cm above the ground. The mouse's activity was recorded on video for 4 minutes, and the cumulative activity time in the last 3 minutes was analyzed (defined as the state in which the mouse was passively suspended and had no limb movement except for respiration).

[0039] Sucrose Preference Experiment (SPT): After the acclimatization period, food and water were withheld for 12 hours. During the test, two pre-weighed bottles were provided to each cage simultaneously: one containing 1.5% (w / v) sucrose solution and the other containing pure water. After 1 hour, the bottles were removed and weighed. Sucrose preference rate = (Sucrose solution consumption / (Sucrose solution consumption + Pure water consumption)) × 100%.

[0040] Table 2. Effects of fermented Lactobacillus mucinus ZD-011 on sucrose preference in depressed mice.

[0041]

[0042] Result: As Figure 2 As shown in Figure 1 and Table 2. Compared with the normal control group, the model group showed significant depressive-like behaviors in all three behavioral tests (p < 0.05). Compared with the model group, the ZD-011 intervention group significantly increased the open field center dwell time by 35.08% ( Figure 2 (A) in the middle), increased the tail activity time by 27.33% ( Figure 2 (B) and increased the sucrose preference index by 25.52% (Table 2) (p < 0.05). This indicates that Lactobacillus fermentum ZD-011 itself has clear antidepressant activity.

[0043] Example 3: Pharmacokinetic Study of Theanine in Mice

[0044] Laboratory animals and drug administration: Healthy male C57BL / 6J mice, 4 weeks old, weighing 12-18 g, were purchased from Shanghai Silex Laboratory Animal Co., Ltd. Animals were acclimatized for one week with free access to food and water, and a 12 / 12-hour light / dark cycle was maintained.

[0045] Drugs and reagents: L-theanine standard and raw materials (purity ≥99%) were purchased from Sangon Biotech (Shanghai) Co., Ltd., product number A504056-0025. Acetonitrile and formic acid were of chromatographic grade.

[0046] Grouping and Administration: Mice were randomly divided into 8 groups of 6 mice each, corresponding to time points of 0 (blank control), 0.5, 1, 2, 4, 8, 16, and 24 hours after gavage. Theanine powder was weighed and prepared into a clear solution of 3 mg / mL with sterile physiological saline. Except for the 0-hour group, which was administered an equal volume of physiological saline by gavage, the other groups of mice were administered theanine solution by gavage at a single dose of 60 mg / kg body weight.

[0047] Sample collection: At each predetermined time point, mice were lightly anesthetized with isoflurane, and approximately 0.5 mL of whole blood was collected from the eyeballs into centrifuge tubes. After the blood was allowed to stand at room temperature for 30 minutes, it was centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant serum was carefully aspirated, aliquoted into EP tubes, and immediately stored in an ultra-low temperature freezer at -80°C.

[0048] Sample pretreatment and LC-MS / MS analysis:

[0049] Serum sample preparation: The collected blood was placed in a 1.5 mL centrifuge tube and allowed to stand at room temperature for 20 minutes. Then, it was centrifuged at 3500 r / min for 15 minutes at 4℃. The upper serum layer was carefully aspirated and immediately transferred to a new centrifuge tube. The sample was then frozen at -80℃ for later use.

[0050] Serum sample pretreatment: Before analysis, thaw frozen serum on ice. Take 50 µL of serum sample and accurately add 200 µL of pre-chilled chromatographic grade acetonitrile. Vortex for 3 minutes to fully precipitate proteins. Then, centrifuge at 15000 g for 15 minutes at 4°C. Accurately pipette 230 µL of the supernatant into a new lyophilization tube and completely remove the solvent using a vacuum freeze dryer. Redissolve the lyophilized residue in 60 µL of ultrapure water, vortex to mix, and centrifuge again at 15000 g for 15 minutes at 4°C. Use the supernatant for LC-MS / MS analysis.

[0051] LC-MS / MS analysis conditions:

[0052] Chromatographic system: Waters ACQUITY UPLC system was used.

[0053] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm).

[0054] Mobile phase: Phase A is ultrapure water containing 0.1% formic acid (FA), and Phase B is acetonitrile (ACN).

[0055] Flow rate: 0.3 mL / min.

[0056] Column temperature: 30℃.

[0057] Injector temperature: 14℃.

[0058] Injection volume: 5 µL.

[0059] Gradient elution program: 0-2.5 min, maintain 5% phase B; 2.5-4.0 min, phase B linearly increases to 95%; 4.0-7.5 min, maintain 95% phase B; 7.5-8.0 min, phase B linearly decreases to 5%; 8.0-10.0 min, maintain 5% phase B to balance the system.

[0060] Mass spectrometry detection conditions:

[0061] Mass spectrometry system: triple quadrupole mass spectrometer detector.

[0062] Ion source: Electrospray ionization (ESI) source, positive ion mode.

[0063] Scanning method: Multiple response monitoring (MRM).

[0064] Detection of ion pair: theanine, m / z 175 → 157.9 (quantitative ion pair).

[0065] Ion source parameters, such as capillary voltage, cone voltage, desolvation gas temperature and flow rate, were determined after optimization to ensure the best response.

[0066] Quantitative analysis: The absolute concentration of theanine in serum samples was calculated using the external standard method based on the theanine standard curve.

[0067] Result: As Figure 3 As shown, the concentration of theanine in mouse serum reached a peak of (17997.46±1980.54) ng / mL (Tmax = 0.5 h) 0.5 h after administration. This result clarifies the basic absorption kinetics of theanine.

[0068] Example 4: Effect of fermentation of Lactobacillus mucilaginosus ZD-011 on the in vivo absorption of theanine

[0069] Objective: To verify the actual effect of strain ZD-011 on the in vivo pharmacokinetics of theanine.

[0070] Experimental animals and grouping: Healthy adult male C57BL / 6J mice were randomly divided into 2 groups (n=6 mice / time point / group):

[0071] Control group (Theanine): Theanine solution (60 mg / kg) was administered by gavage.

[0072] Experimental group (Theanine + ZD-011): Oral administration of theanine (60 mg / kg) and a suspension of *Lactobacillus mucinus* ZD-011 (2 × 10⁻⁶) 8 A mixture of CFU / unit (total volume 200 µL).

[0073] Both groups of mice were pre-treated by daily gavage with the corresponding liquid (physiological saline for the control group and ZD-011 bacterial suspension for the experimental group) for 7 consecutive days to adapt to the intervention.

[0074] Sample collection: On day 8, after both groups of mice were administered the corresponding drug (theanine or theanine + bacteria) by gavage in the morning, blood samples were collected at eight time points: 0 (pre-administration blank), 0.5, 1, 2, 4, 8, 16, and 24 hours. A separate group of mice was used for each time point (to avoid interference from repeated sampling). Blood sample processing and serum preparation methods were the same as in Example 3.

[0075] Theanine concentration detection: The concentration of theanine in serum was detected using the same LC-MS / MS method as in Example 3.

[0076] Data Processing and Results: The average serum theanine concentration at each time point was calculated for both groups of mice. Drug-time curves were plotted (e.g.,...). Figure 4 (as shown), and the main pharmacokinetic parameters were calculated.

[0077] (1) Drug-time curve analysis: Compared with the control group, the serum theanine concentration-time curve of the experimental group (theanine + ZD-011) shifted significantly upward. The concentration of theanine in the experimental group was significantly higher than that in the control group at the peak time (0.5 hours) and in the following 3-4 hours.

[0078] (2) Comparison of key parameters: The main pharmacokinetic parameters of the experimental group, such as the area under the curve (AUC0-24h, reflecting the total exposure) and peak concentration (Cmax), were significantly higher than those of the control group (p<0.05).

[0079] Conclusion: This embodiment confirms from a pharmacokinetic perspective that fermentation of *Lactobacillus myxitis* ZD-011 can consistently and significantly increase the in vivo exposure level of theanine. This provides a direct and robust pharmacokinetic explanation for the subsequently observed behavioral synergistic antidepressant effect.

[0080] Example 5: Verification of the synergistic antidepressant effect of fermented Lactobacillus mucinus ZD-011 and theanine.

[0081] The method for establishing the depression model is the same as in Example 2.

[0082] Experimental grouping and intervention: Mice were randomly divided into the following 6 groups, with 6 mice in each group:

[0083] Table 3. Animal experimental groupings for enhancing the antidepressant effect of Lactobacillus mucinus ZD-011.

[0084]

[0085] Behavioral tests: Sucrose preference, tail suspension, and open field tests were conducted on days 30-35 of the experiment.

[0086] Results analysis: All data are expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons, and p < 0.05 was considered statistically significant.

[0087] Result: As Figure 5 As shown.

[0088] Model validation was successful: Compared with the normal control group, the model group (CORT) mice exhibited typical depressive-like behaviors: significantly reduced sucrose preference rate (anhedonia), significantly prolonged tail immobility time (behavioral despair), and significantly reduced time spent in the central area of ​​the open field and distance traveled (decreased exploration willingness and anxiety-like behaviors) (all p < 0.05).

[0089] Figure 5 In the open field experiment (A), compared to the normal control group, the central dwell time in the model group was significantly reduced by 36.77%. The positive control group, theanine group, and ZD-011 alone all significantly improved this behavior. Most importantly, the combined intervention group (theanine + ZD-011) showed the most significant improvement, increasing by 68.63%, with its central dwell time not only fully recovering to normal levels but also showing a trend superior to the positive control group and any single component group.

[0090] Figure 5 (B) Tail suspension experiment: The immobility time in the model group was significantly increased. This phenomenon was significantly reversed in the theanine group and the positive control group (p < 0.05 relative to the model group). The ZD-011 strain alone showed some improvement. The combined intervention group showed a clear anti-despair effect.

[0091] Figure 5 (C) Sucrose preference experiment: The sucrose preference rate in the model group was significantly reduced. The positive drug group, theanine group, strain ZD-011 alone group and combined intervention group all significantly improved the sucrose preference rate, restoring it to the normal level (p < 0.05 relative to the model group), and there was no significant difference among the groups, indicating that they were all effective in improving the symptoms of core anhedonia.

[0092] Conclusion: The above behavioral results indicate that both *Lactobacillus fermentum* ZD-011 and theanine possess clear antidepressant activity. When used in combination, they exhibit a synergistic effect, exceeding that of either ingredient, particularly in improving exploratory motivation and anxiety-like behaviors (open field test).

Claims

1. A fermenting *Lactobacillus mucinus*, characterized in that, Classified as Lactobacillus fermentans Limosilactobacillus fermentum , Plant No. ZD-011, Preservation No. CCTCC NO: M 20251136.

2. The application of the fermented *Lactobacillus mucinus* according to claim 1 in the preparation of antidepressant products, characterized in that, Antidepressant products can be medicines or animal feed.

3. A composition, characterized in that, It contains L-theanine and the fermented myxobacterium as described in claim 1.

4. The composition according to claim 3, characterized in that, Each 1.2 mg of L-theanine corresponds to 1 × 10 7 ~1×10 11 The CFU contains live fermented *Lactobacillus mucinus* bacteria.

5. The composition according to claim 3, characterized in that, It also contains pharmaceutically acceptable carriers and / or prebiotics.

6. The composition according to claim 3, characterized in that, Dosage forms include powder, granules, capsules, tablets, or oral liquid.

7. The use of the composition according to any one of claims 3 to 6 in the preparation of an antidepressant product, characterized in that, Antidepressant products can be medicines or animal feed.

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