Plant lactobacillus ZD-012, composition and application

Abstract

The application discloses a plant lactobacillus ZD-012, a composition and application. The plant lactobacillus ZD-012 is classified as plant lactobacillus Lactiplantibacillus plantarum , with a strain number ZD-012 and a preservation number CGMCC No. 34613. The strain has a clear function of promoting theosine absorption, and does not show obvious direct antidepressant activity. It is disclosed that the ZD-012 enhances theosine efficacy through a single and clear mechanism of promoting absorption-increasing exposure. This provides a brand-new and mechanism-identified probiotic resource for developing an efficiency-enhancing antidepressant product.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of Lactobacillus plantarum ZD-012, its composition, and its application. Background Technology

[0002] The demand for treatment of depression is enormous, and natural, safe alternatives are receiving considerable attention. Theanine, a natural amino acid with antidepressant potential, suffers from high oral bioavailability, a key bottleneck limiting its stable efficacy. Improving the bioavailability of theanine is therefore an important strategy for enhancing its therapeutic effects.

[0003] Some plant lactobacillus (Lactobacillus plantarum) have been reported in the prior art. Lactiplantibacillus plantarum It has its own antidepressant effect.

[0004] For example, the invention application with publication number CN120041355A discloses a highly active plant lactobacillus KL100 for relieving anxiety and depression and its uses. This plant lactobacillus KL100 has a good gastrointestinal pass rate. After being encapsulated, it exhibits reproductive characteristics in the stomach, improves bile salt tolerance and survival rate, can effectively reduce the expression of IL-1β and TNF-α in mice, increase the expression of IL-10, and promote the secretion of 5-hydroxytryptamine by intestinal chromaffin cells, thereby relieving anxiety and depression.

[0005] The invention application with publication number CN119931874A discloses a strain of *Lactobacillus plantarum* GOLDGUT-HNU082 and its applications. GOLDGUTHNU082 can significantly alleviate depressive-like behaviors in mice induced by chronic, unpredictable, mild stress, including reducing anxiety and despair behaviors and increasing pleasure, thus improving depression; it significantly increases the levels of key neurotransmitters such as serotonin (5HT), dopamine (DA), gamma-aminobutyric acid (GABA), and norepinephrine (NE) in the colon, serum, and brain, regulating emotional responses; it significantly reduces the levels of interleukin-6 (IL6), tumor necrosis factor-α (TNFα), and lipopolysaccharide (LPS), alleviating inflammatory responses; it reduces neuronal damage in the brain; and it improves intestinal flora imbalance in depressed mice, reducing harmful bacteria and increasing the abundance of beneficial bacteria.

[0006] Probiotics, as an important component of the gut microbiota, can influence the body's physiological state by regulating intestinal barrier function and promoting nutrient absorption. Existing studies have shown that some Lactobacillus strains can promote the absorption of amino acids in the gut. However, there is a lack of research on targeted screening of probiotic strains that specifically enhance the absorption of theanine, based on key pharmacokinetic parameters of theanine, and that do not rely on significant antidepressant activity to exert their synergistic effect. This kind of strain screening and application aimed at "precise synergistic effect" represents a technological gap and an innovative direction in this field. Summary of the Invention

[0007] To address the aforementioned shortcomings in the prior art, this invention provides a strain of *Lactobacillus plantarum* ZD-012, a composition thereof, and its applications.

[0008] This invention first provides a strain of *Lactobacillus plantarum* ZD-012, classified and named *Lactobacillus plantarum*. Lactiplantibacillus plantarum , Plant No. ZD-012, Collection No. CGMCC No. 34613.

[0009] This invention further provides the application of *Lactobacillus plantarum* ZD-012 in the preparation of antidepressant products, wherein *Lactobacillus plantarum* ZD-012 is used in combination with theanine. Preferably, the antidepressant product is a health food, pharmaceutical, or animal feed.

[0010] The present invention also provides a composition comprising theanine and the aforementioned Lactobacillus plantarum ZD-012.

[0011] Preferably, the theanine dosage is 20-100 mg / kg body weight. More preferably, the theanine dosage is 60 mg / kg body weight. Each 1.2 mg of theanine corresponds to a viable bacterial count of 2 × 10⁻⁶. 8 The CFU is the plant lactobacillus ZD-012.

[0012] Preferably, the composition further comprises a pharmaceutically or food-grade acceptable carrier and / or prebiotic.

[0013] The present invention also provides the use of the composition in the preparation of antidepressant products. Preferably, the antidepressant product is a health food, a pharmaceutical, or animal feed.

[0014] This invention first precisely determined the pharmacokinetics of theanine in mice, identifying that its serum concentration peaked 0.5 hours after gavage. Using this critical time to peak (Tmax) as the sole screening node, a candidate bacterial library containing various lactobacilli was targeted for screening. The results showed that *Lactobacillus plantarum* ZD-012 significantly increased serum theanine concentration at this time point.

[0015] Further systems pharmacokinetic experiments confirmed that when strain ZD-012 was co-treated with theanine, it could significantly and persistently increase the total exposure (AUC) of theanine in serum.

[0016] Ultimately, in a corticosterone-induced depression model, it was verified that strain ZD-012 alone did not significantly improve depressive behavior, but when used in combination with theanine, it significantly enhanced the antidepressant effect of theanine, demonstrating a clear synergistic effect.

[0017] The beneficial effects of this invention are as follows: 1. A functionally specific strain of Lactobacillus plantarum ZD-012 was obtained. This strain has a clear function of "promoting theanine absorption" and does not show obvious direct antidepressant activity.

[0018] 2. This study revealed the mechanism by which ZD-012 enhances the efficacy of theanine through a single, clear mechanism of "promoting absorption-increasing exposure." This provides a novel, well-defined probiotic resource for developing "synergistic" antidepressant products.

[0019] 3. A feasible approach is provided for screening "absorption-enhancing" functional probiotics based on key in vivo pharmacokinetic parameters (Tmax).

[0020] 4. The strains and compositions described herein are all derived from natural sources, have high safety and no side effects, and have higher safety and tolerability compared to existing clinical antidepressants (such as fluoxetine), making them suitable for long-term use. Attached Figure Description

[0021] Figure 1 Example 1: The time-dependent curve of serum theanine concentration in mice after oral administration of theanine (60 mg / kg) is shown.

[0022] Figure 2 Example 2: A bar chart comparing the theanine content in the serum of mice in different candidate probiotic intervention groups 0.5 hours after oral administration of theanine. * indicates p < 0.05 compared to the control group.

[0023] Figure 3 Example 3: 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 + Lactobacillus plantarum ZD-012) over time.

[0024] Figure 4 Example 4 illustrates the synergistic effect of *Lactobacillus plantarum* ZD-012 combined with theanine on behavioral indicators in a depressed mouse model. Among other things, Figure 4 (A) in the middle: Open field experiment; Figure 4 (B) in the figure: Tail suspension test. # indicates p < 0.05 compared with the normal group, #### indicates p < 0.0001 compared with the normal group, * indicates p < 0.05 compared with the model group, ** indicates p < 0.01 compared with the model group, **** indicates p < 0.0001 compared with the model group. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the methods used in the following embodiments are conventional methods, and the reagents and materials used are commercially available.

[0026] Example 1: Pharmacokinetic Study of Theanine in Mice Experimental animals and drug administration: Healthy C57BL / 6J male mice (n=6 mice / time point) were administered a single oral gavage of theanine solution at a dose of 60 mg / kg body weight (theanine was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number A504056-0025, and prepared with physiological saline to a concentration of 3 mg / mL).

[0027] Sampling and detection: Serum was collected at 0 (blank), 0.5, 1, 2, 4, 8 and 12 hours after drug administration. The concentration of theanine was determined by LC-MS / MS (chromatographic column: Waters ACQUITY BEH C18; mobile phase: 0.1% formic acid water-acetonitrile; detection ion pair: m / z 175→157.9).

[0028] Result: As Figure 1 As shown, the concentration of theanine in mouse serum reached a peak of (16892.53±2358.38) ng / mL (Tmax = 0.5 h) 0.5 h after administration, and then gradually decreased. At 12 h, the serum theanine content dropped to near the detection limit, and the sampling time for subsequent experiments was determined to be 0.5 h after gavage.

[0029] Example 2: Screening of probiotic strains that promote theanine absorption Candidate strains: Six Lactobacillus strains with potential regulatory functions were selected from the probiotic resource bank of our laboratory. These strains were isolated from fecal samples of healthy volunteers, traditionally fermented fruits and vegetables / dairy products, and standard probiotic strains donated by relevant research partners. These included: *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum LP01 and ZD-012, Lactobacillus fermentum ( Limosilactobacillus fermentum LF01 and LF02, Lactobacillus salivarius ( Ligilactobacillus salivarius LS01 and LS02.

[0030] Preparation of bacterial suspension: All candidate strains were inoculated into MRS liquid medium and cultured aerobically at 37°C until the late logarithmic phase. The bacterial cells were collected by centrifugation, resuspended in sterile physiological saline, and the concentration was adjusted to 1×10⁻⁶. 9 CFU / mL.

[0031] Experimental design and grouping: C57BL / 6J mice were randomly divided into 8 groups, with 6 mice in each group: blank control group (administered by gavage with physiological saline), theanine alone group, and 6 "theanine + candidate bacteria" groups.

[0032] Intervention and Sampling: The bacterial intervention group was given 0.2 mL of the corresponding bacterial suspension (containing 2×10⁻⁶ bacteria) by gavage daily. 8Live CFU (cytokine FU) was administered for 7 consecutive days. The control group was given an equal volume of physiological saline by gavage. On day 7 of the intervention, 30 minutes after gavage administration of the bacterial solution (or physiological saline), all mice except the blank control group were given a theanine solution at a dose of 60 mg / kg by gavage. Serum was collected precisely 0.5 hours after the administration of theanine.

[0033] Theanine concentration detection: After protein precipitation in acetonitrile and centrifugation, the supernatant of serum samples was collected and the theanine concentration was detected using the same LC-MS / MS method as in Example 1.

[0034] Result: As Figure 2 As shown, compared with the theanine alone (17379.98±1820.97) ng / mL, only the intervention of *Lactobacillus plantarum* ZD-012 significantly increased the serum theanine concentration in mice by 65.15% (p<0.05). No statistically significant increase was observed when other candidate strains (LP01, LF01, LF02, LS01, LS02) were used in combination with theanine (p>0.05). ZD-012 was identified as a positive strain with specific theanine uptake-promoting function.

[0035] Example 3: Effect of Lactobacillus plantarum ZD-012 on in vivo exposure of theanine Experimental animals and grouping: Healthy adult male C57BL / 6J mice were randomly divided into 2 groups (n=6 mice / time point / group): Control group (Theanine): administered normal saline by gavage daily for 7 consecutive days; on the 8th day, administered theanine solution (60 mg / kg) by gavage.

[0036] Experimental group (Theanine + ZD-012): Daily gavage administration of *Lactobacillus plantarum* ZD-012 bacterial suspension (2 × 10⁻⁶) 8 CFU / animal), for 7 consecutive days; on the 8th day, theanine (60 mg / kg) and ZD-012 bacterial suspension (2×10) were administered by gavage. 8 A mixture of CFU (total volume 200 µL).

[0037] Sample collection and testing: Blood samples were collected at 0 (before administration), 0.5, 1, 2, 4, 8, and 12 hours after gavage on day 8 to prepare serum. Theanine concentration was detected using the same LC-MS / MS method as in Example 1.

[0038] Data processing and results: Plotting the drug-time curve as follows Figure 3 As shown.

[0039] (1) Drug-time curve analysis: Compared with the control group, the serum theanine concentration-time curve of the experimental group (theanine + ZD-012) shifted significantly upward. At the peak time of theanine (0.5 hours) and in the following 1-2 hours, the concentration of the experimental group was significantly higher than that of the control group (p<0.05).

[0040] (2) Comparison of key parameters: The area under the curve (AUC0-12h, reflecting total exposure) and peak concentration (Cmax) of the experimental group were significantly higher than those of the control group (p<0.05). This indicates that strain ZD-012 can significantly improve the systemic exposure level and absorption efficiency of theanine in vivo.

[0041] Example 4: Verification of the effect of Lactobacillus plantarum ZD-012 on enhancing theanine's antidepressant effect Establishment of the depression model: A mouse depression model induced by chronic corticosterone drinking water was used. Corticosterone (CAS: 50-22-6) was dissolved in an aqueous solution containing 0.45% (w / v) hydroxypropyl-β-cyclodextrin (HP-β-CD) to prepare HP-β-CD water with a corticosterone concentration of 0.1 mg / mL. The mice were allowed free access to the water, and the daily intake was controlled at approximately 5 mg / kg body weight for 4 weeks.

[0042] Experimental grouping and intervention: As shown in Table 1, C57BL / 6J mice were randomly divided into the following 5 groups, with 6 mice in each group.

[0043] Table 1. Animal experimental groupings for enhancing the antidepressant effect of Lactobacillus plantarum ZD-012 and theanine.

[0044] Behavioral tests: The sugar preference test (SPT), the suspended tail test (TST), and the open field test (OFT) were conducted in the fourth week of continuous intervention.

[0045] Result: As Figure 4 As shown in Table 2.

[0046] (1) Model validation: Consistent with literature reports and expectations, the model group exhibited typical depressive-like behaviors (reduced sucrose preference, prolonged tail immobility time, and reduced open field exploration).

[0047] (2) The positive control group (fluoxetine) showed the expected antidepressant effect.

[0048] (3) Theanine alone: ​​Theanine alone significantly improved depressive behavior (p<0.05 compared with the model group), verifying the antidepressant effect of theanine.

[0049] (4) ZD-012 alone: ​​Compared with the model group, the ZD-012 alone group showed no significant improvement in any of the three behavioral indicators (p>0.05), indicating that Bacillus plantarum ZD-012 itself does not have significant direct antidepressant activity.

[0050] (5) Key synergistic effect: The combined group (theanine + ZD-012) showed significantly better improvement in all behavioral tests than the theanine alone group (p < 0.05). Compared with the model group, the combined use of theanine and ZD-012 prolonged the tail suspension activity time by 65.63%, increased the dwell time in the central region of the open field by 68.63%, and improved the sucrose preference rate by 30.75%. This indicates that *Lactobacillus plantarum* ZD-012 can effectively enhance the antidepressant effect by promoting the absorption of theanine, thus achieving the synergistic effect.

[0051] Table 2. Effects of combined use of *Lactobacillus plantarum* ZD-012 and theanine on sucrose preference in a depressed mouse model.

[0052] Example 5: Identification of strain ZD-012 Morphological and biochemical characteristics: When strain ZD-012 was cultured aerobically at 37°C on MRS agar plates, it formed milky white, round, raised colonies with neat edges.

[0053] Molecular biological identification: Genomic DNA was extracted from the strain, its 16S rRNA gene sequence was amplified and sequenced. BLAST comparison with the NCBI database showed it to be similar to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The sequence similarity of the model strain exceeded 99%. The 16S rRNA sequence of the strain is as follows:

[0054] Strain preservation information: This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 21, 2025, and is classified as *Lactobacillus plantarum*. Lactiplantibacillus plantarum , Plant No. ZD-012, Collection No. CGMCC No. 34613.

Claims

1. A strain of *Lactobacillus plantarum* ZD-012, characterized in that, Classified as Lactobacillus plantarum Lactiplantibacillus plantarum , Plant No. ZD-012, Collection No. CGMCC No. 34613.

2. The use of Lactobacillus plantarum ZD-012 according to claim 1 in the preparation of antidepressant products, wherein Lactobacillus plantarum ZD-012 is used in combination with theanine.

3. The application according to claim 2, characterized in that, Antidepressant products can be health foods, medicines, or animal feed.

4. A composition, characterized in that, It contains theanine and the Lactobacillus plantarum ZD-012 as described in claim 1.

5. The composition according to claim 4, characterized in that, The dosage of theanine is 20-100 mg / kg body weight, and each 1.2 mg of theanine corresponds to a viable bacterial count of 2 × 10⁻⁶. 8 The CFU is the plant lactobacillus ZD-012.

6. The composition according to claim 4, characterized in that, It also contains pharmaceutically or food-grade acceptable carriers and / or prebiotics.

7. Use of the composition according to any one of claims 4 to 6 in the preparation of an antidepressant product.

8. The application according to claim 7, characterized in that, Antidepressant products can be health foods, medicines, or animal feed.

Images