Lactobacillus plantarum KLDS1.0344 as well as preparation and application thereof
By providing the Lactobacillus plantarum KLDS1.0344 strain and its bacterial suspension, the problem of limited antidepressant strain resources in the prior art is solved, achieving multi-dimensional improvement of depressive symptoms. It provides a complete chain solution from strain to multi-dosage form composition, highlighting the systemic advantages.
Patent Information
- Application Number
- CN202610044953.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-14
- Publication Date
- 2026-02-24
AI Technical Summary
The resources of Lactobacillus plantarum strains with clear antidepressant effects in the current technology are limited, and there is a lack of systematic dosage and mechanism studies to verify their efficacy, which leads to limited clinical treatment compliance and long-term prognosis.
We provided a strain of Lactobacillus plantarum KLDS1.0344 and its bacterial suspension. Through systematic experiments, we verified its effective dose range and multi-dimensional improvement effects in whole animal models, including the regulation of behavior, neuroendocrine and gut microbiota. We constructed a complete chain solution from strain to multi-dosage formulation composition.
It significantly improves depressive symptoms. Animal experiments have verified the multidimensional effects of the strain on behavior, neuroendocrine function, and gut microbiota, providing a safe and effective microbial resource and offering new solutions for the development of antidepressant drugs, health foods, and general foods.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology and applications, specifically to a strain of Lactobacillus plantarum KLDS1.0344 that has the effect of improving depression, as well as preparations containing this strain and their use in improving depressive symptoms or alleviating depressive mood. Background Technology
[0002] Depression is a common mental disorder characterized by persistent low mood and anhedonia, often accompanied by cognitive impairment. Its global incidence continues to rise, resulting in a heavy physical and social burden. Currently, first-line clinical treatment mainly relies on chemotherapeutic agents such as selective serotonin reuptake inhibitors (SSRIs). However, these drugs generally have limitations, including slow onset of action, large individual variability in response rates, numerous side effects, and high relapse rates after discontinuation, severely impacting patient adherence and long-term prognosis. Therefore, developing safe and novel adjunctive or alternative intervention strategies is of significant clinical importance.
[0003] With the deepening of microbiology research, the regulatory potential of specific probiotics on host mood and behavior has attracted increasing attention. Studies have shown that some probiotic strains may have a positive impact on depressive-like behaviors through pathways such as regulating immune inflammation, affecting neuroendocrine function, and central neurotransmitter metabolism. Among them, *Lactobacillus plantarum* is considered an important resource for developing strains with mood-regulating functions due to its long history of food safety applications, good gastrointestinal adaptability, and diverse metabolic characteristics. However, the resources of *Lactobacillus plantarum* strains with clear and stable antidepressant efficacy are still relatively limited, and most studies only examine single or a few indicators, lacking sufficient evidence for systematic correlation and synergistic validation of behavioral, neuroendocrine, and key neurochemical indicators in whole animal models.
[0004] Therefore, there is an urgent need in this field to obtain novel strains of Lactobacillus plantarum that have undergone rigorous functional validation, and to provide systematic efficacy data and mechanism of action in whole animal models, in order to fill the gaps in existing technologies and provide new microbial resources and solutions for the development of related products for improving depressive symptoms or mood. Summary of the Invention
[0005] In view of the limited resources of Lactobacillus plantarum strains with clear antidepressant effects in the existing technology, and the lack of systematic dosage and mechanism studies to verify their efficacy, the present invention provides a Lactobacillus plantarum strain KLDS1.0344 with clear efficacy in improving depression and which has been systematically verified.
[0006] Furthermore, the present invention aims to provide a bacterial suspension containing the above-mentioned strains and with a defined effective dosage range.
[0007] Furthermore, the present invention aims to provide a microbial composition that uses the above-mentioned strains or their bacterial suspensions as active ingredients and can be used in various dosage forms.
[0008] Furthermore, the present invention also aims to provide a method for preparing the above-mentioned bacterial suspension.
[0009] Furthermore, the present invention aims to provide a clear use of the above-mentioned strains, bacterial suspensions or compositions in the preparation of products for improving depressive symptoms or mood.
[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0011] Firstly, provide a strain of Lactobacillus plantarum ( Lactobacillus plantarum KLDS1.0344, with accession number CCTCC NO: M 2022803.
[0012] Secondly, a bacterial suspension is provided, which contains the aforementioned bacterial strains and has a viable cell concentration of 1×10⁻⁶. 7 CFU / mL to 1×10 9 CFU / mL.
[0013] Thirdly, a microbial composition is provided, wherein the above-mentioned strain, or its cells, or the above-mentioned bacterial suspension, is used as an active ingredient.
[0014] Fourthly, a method for preparing the bacterial suspension is provided, comprising the steps of activating the bacterial strain, subculturing, collecting the bacterial cells, and resuspending them.
[0015] Fifthly, the use of the strain, bacterial suspension, or composition in the preparation of articles for improving depressive symptoms or alleviating depressive mood is provided.
[0016] Preferably, the microbial composition can be prepared as food, health food, or medicine.
[0017] Preferably, the product used in the intended purpose is an antidepressant.
[0018] Compared to existing technologies, the beneficial effects of this invention lie in its first disclosure of *Lactobacillus plantarum* KLDS1.0344, which has been well-preserved and has been verified through systematic in vivo experiments to possess significant antidepressant efficacy. A complete supply chain solution has been constructed, encompassing the strain, bacterial suspension, and multi-dosage form compositions, along with preparation methods and clearly defined uses. Animal experiments not only determined the effective dosage range but also systematically verified its multi-dimensional effects on behavior, neuroendocrine function, neurochemistry, and gut microbiota. From a "microbe-gut-brain axis" perspective, it provides a more comprehensive and in-depth mechanistic explanation than existing technologies, highlighting the systematic advantages of this invention. This provides a safe and effective new microbial resource for the development of antidepressant drugs, health foods, and general foods. Attached Figure Description
[0019] Figure 1 This is a graph showing the statistical results of the movement trajectory, total movement distance, and immobility time of each group of mice in the open field test;
[0020] Figure 2 This is a graph showing the statistical results of the immobility time of mice in each group during the tail suspension test;
[0021] Figure 3 This is a graph showing the statistical results of the immobility time of each group of mice during the forced swimming test;
[0022] Figure 4 This is a graph showing the statistical results of the sugar water preference index of each group of mice in the sugar water preference test;
[0023] Figure 5 This is a graph showing the statistical results of serum levels of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone in each group of mice.
[0024] Figure 6 This is a graph showing the statistical results of dopamine levels in the serum of mice in each group;
[0025] Figure 7 This is a graph showing the statistical results of norepinephrine levels in the serum of mice in each group;
[0026] Figure 8 This is a graph showing the statistical results of 5-hydroxytryptamine in the hippocampus of mice in each group;
[0027] Figure 9 This is a graph showing the statistical results of brain-derived neurotrophic factor in the hippocampus of mice in each group;
[0028] Figure 10 This is a graph showing the statistical results of the gut microbiota chao1 and observed_species in mice from the NC, MC, and HP groups;
[0029] Figure 11 This is a graph showing the statistical results of the changes in the abundance of gut microbiota at the phylum and genus levels in mice from the NC, MC, and HP groups.
[0030] In each figure, * p<0.05, **p<0.01 indicates statistical significance compared with the NC group; #p<0.05, ##p<0.01 indicates statistical significance compared with the MC group. Detailed Implementation
[0031] The technical solutions and effects of the present invention will be clearly and completely described below with reference to specific embodiments and accompanying drawings. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solutions of the present invention and should not be regarded as limiting the scope of protection of the present invention. Unless otherwise specified, the test methods used in the following embodiments are conventional methods; unless otherwise specified, the raw materials used are items commonly used in the art, publicly available, or commercially obtainable.
[0032] The preservation information involved in this invention is as follows: Preservation name: Lactobacillus plantarum KLDS1.0344; Strain preservation number: CCTCC NO: M2022803; Date of preservation: June 7, 2022.
[0033] In the following examples, the MRS medium was prepared as follows: 5.0 g of peptone, 10.0 g of tryptone, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 20.0 g of glucose, 5.0 g of yeast extract, 0.25 g of manganese sulfate, 0.58 g of magnesium sulfate, 2.0 g of diammonium hydrogen citrate, 1.0 g of Tween-80, and 5.0 g of beef extract were dissolved in distilled water and brought to a final volume of 1 L. The mixture was stirred thoroughly and sterilized at 121°C for 15 min to obtain the MRS medium. In other examples, the MRS medium can be obtained commercially available.
[0034] The technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0035] Example 1: Preparation of Lactobacillus plantarum KLDS1.0344 bacterial suspension
[0036] The cryovial of *Lactobacillus plantarum* KLDS1.0344 (accession number CCTCC NO: M 2022803) was removed from the -80°C freezer and thawed on ice. In an aseptic environment, the inoculum was transferred at a 2% (v / v) inoculation rate to a test tube containing 10 mL of MRS liquid medium and mixed thoroughly. The test tube was then placed in a 37°C anaerobic incubator and incubated statically for 24 hours to complete the first activation.
[0037] Take the activated bacterial culture and transfer it to fresh MRS liquid medium at an inoculum rate of 2% (v / v). Continue culturing under the same conditions (37°C, anaerobic) for 24 hours to complete the second activation and subculturing. Repeat this step once more, that is, perform two subculturings in total, to obtain stable and highly active bacterial cells.
[0038] The final cultured bacterial suspension was centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was discarded. The bacterial sludge was gently washed with pre-cooled 0.9% (w / v) sterile physiological saline, centrifuged again, and washed twice more to thoroughly remove culture medium components. Finally, the bacterial sludge was resuspended in an appropriate amount of sterile physiological saline, and the concentration was determined using the plate count method. Three bacterial suspensions of low, medium, and high concentrations were prepared by dilution for later use, with viable cell concentrations of approximately 1 × 10⁻⁶. 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL. The prepared bacterial suspension was stored at 4°C and used for subsequent animal experiments within 24 hours.
[0039] Example 2: Study on the ameliorative effect and mechanism of Lactobacillus plantarum KLDS1.0344 on depressive-like behavior in CUMS mice.
[0040] 1. Establishment of experimental animals and models
[0041] Healthy 5-week-old male BALB / c mice were selected and, after one week of acclimatization, randomly divided into 5 groups (n=12): blank control group (NC), model control group (MC), low-dose KLDS1.0344 group (LP), medium-dose KLDS1.0344 group (MP), and high-dose KLDS1.0344 group (HP). Except for the blank control group, all other groups underwent chronic unpredictable mild stress to establish a depression model. A stressor was randomly applied daily (restraint, reversed day / night cycle, fasting, water deprivation, horizontal shaking, hypothermic swimming, tail clamping, damp bedding, tilted cage, stroboscopic light, lack of bedding, odor, overcrowding) for 5 weeks. From the first week of modeling, all treatment groups received daily gavage intervention at the same time. Each KLDS1.0344 dose group was administered the corresponding concentration of bacterial suspension prepared in Example 1, with a gavage volume of 0.2 mL / mouse / day. The blank control group and model group were administered an equal volume of physiological saline. Administered once daily until the end of the experiment.
[0042] After the modeling and intervention are completed, the following behavioral tests will be conducted. All behavioral tests will be conducted in a quiet environment with uniform lighting.
[0043] 2. Behavioral tests
[0044] (1) Open field test: The mouse was gently placed in the center of the open field test box (50 cm × 50 cm × 50 cm black box) and allowed to explore freely for 5 minutes. The video system recorded the total distance it moved and the time it remained stationary.
[0045] The results are as follows Figure 1As shown, compared with the NC group, the total movement distance of mice in the MC group was significantly reduced, and the immobility time was significantly prolonged (p<0.01). The total movement distance of mice in the MP and HP groups was significantly increased compared with the MC group (p<0.01), and the immobility time was also significantly shortened (p<0.01), indicating that their spontaneous activity and exploratory inhibition were alleviated.
[0046] (2) Tail suspension test: The tail of the mouse was fixed and suspended with medical tape, so that its head was about 15 cm from the bottom of the box. The test lasted for 6 minutes, and the cumulative immobility time of the body within the last 4 minutes was recorded.
[0047] The results are as follows Figure 2 As shown, the immobility time of mice in the MC group was significantly longer than that in the NC group (p<0.01). Compared with the MC group, the immobility time of mice in the MP and HP groups was significantly shorter (p<0.01), indicating that the behavioral despair state was effectively improved.
[0048] (3) Forced swimming test: The mice were placed in a transparent cylindrical container (15 cm in diameter and 30 cm in height) filled with clean water (25 ± 1°C, 15 cm deep). The test lasted for 6 minutes, and the cumulative immobility time in the last 5 minutes was recorded. The water was changed after each mouse was tested.
[0049] The results are as follows Figure 3 As shown, the immobility time of mice in the MC group was significantly longer than that in the NC group (p<0.01). Compared with the MC group, the immobility time of mice in the MP and HP groups was significantly shorter (p<0.01), indicating that the behavioral despair state was effectively improved.
[0050] (4) Sugar water preference test: The mice were fasted and deprived of water for 12 hours before the test. During the test, the mice were kept in individual cages and had free access to two pre-weighed liquids (one bottle of 1% sucrose solution and one bottle of purified water). The bottles were switched after 6 hours, and the total test time was 12 hours. The mice were weighed again after the test and the sugar water preference rate was calculated (sucrose solution consumption / total liquid consumption × 100%).
[0051] The results are as follows Figure 4 As shown, the sucrose preference rate in the MC group was significantly lower than that in the NC group (p<0.01). The sugar water preference index in the MP and HP groups was significantly different from that in the MC group (p<0.01), indicating a significant improvement in the loss of pleasure.
[0052] 3. Sample collection and biochemical index determination
[0053] Following behavioral testing, mice were anesthetized, and blood was collected via the abdominal aorta. After the blood was allowed to stand at room temperature for 2 hours, it was centrifuged at 3000×g for 10 minutes at 4°C to separate the serum, which was then stored at -80°C. Intestinal contents were collected under aseptic conditions. Bilateral hippocampal tissue was then rapidly separated, flash-frozen in liquid nitrogen, and stored at -80°C. The concentrations of corticotropin-releasing hormone, adrenocorticotropic hormone, corticosterone, dopamine, and norepinephrine in the serum, as well as the levels of serotonin and brain-derived neurotrophic factor in the hippocampus, were determined strictly according to the instructions of the commercial ELISA kit.
[0054] The results of the biochemical index measurements are as follows:
[0055] (1) Adjustment effect on HPA axis function: such as Figure 5 As shown, compared with the NC group, the serum levels of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone were significantly elevated in the MC group (p<0.01). The MP and HP groups significantly reversed the abnormal elevation of these hormones (p<0.01), indicating that KLDS1.0344 can improve CUMS-induced overactivation of the HPA axis.
[0056] (2) Regulatory effects on the levels of monoamine neurotransmitters and brain-derived neurotrophic factor: such as Figure 6 , Figure 7 , Figure 8 and Figure 9 As shown, serum dopamine and norepinephrine levels in the MC group were significantly lower than those in the NC group (p<0.01). Dopamine and norepinephrine levels were significantly elevated in the LP group (p<0.05), and extremely significantly elevated in the MP and HP groups (p<0.01). Hippocampal serotonin and brain-derived neurotrophic factor (BDNF) levels in the MC group were significantly lower than those in the NC group (p<0.01). Serotonin levels in the LP, MP, and HP groups were all extremely significantly higher than those in the MC group (p<0.01), and BDNF levels in the MP and HP groups were also extremely significantly higher (p<0.01).
[0057] 4. Gut microbiota 16S rRNA gene sequencing analysis
[0058] The sequencing method is as follows:
[0059] Colonic contents samples were collected from mice in the NC, MC, and HP groups for 16S rRNA amplicon sequencing. Specifically, after extracting genomic DNA from the samples, PCR amplification was performed targeting the hypervariable region of the bacterial 16S rRNA gene. Libraries were constructed and sequenced using the Illumina platform. The raw data were preprocessed, including quality control and chimera removal, to obtain valid sequences. Operational taxonomic unit clustering and species classification annotation were then performed.
[0060] The sequencing results and analysis are as follows:
[0061] (1) such as Figure 10 As shown, alpha diversity analysis revealed that compared to the NC group, the Chao1 index and Observed_species index of the gut microbiota in the MC group were significantly decreased (p<0.01), indicating that CUMS modeling led to a decrease in gut microbiota richness. The Chao1 index in the HP group was significantly higher than that in the MC group (p<0.01), and the Observed_species index also showed an upward trend. This suggests that high-dose intervention with KLDS1.0344 can effectively reshape the gut microbiota structure of CUMS mice and exert a positive regulatory effect on its species richness.
[0062] (2) such as Figure 11 As shown, based on species composition analysis, at the phylum level, Firmicutes (… Firmicutes ) and Bacteroidetes ( Bacteroidetes Bacteroidetes were the dominant phylum in each group. Compared with the NC group, the MC group had a higher prevalence of Bacteroidetes. Bacteroidetes The relative abundance of [unspecified species] decreased, while its abundance rebounded in the HP group. In addition, [unspecified species] were also detected. Desulfobacterota ), Actinobacteria ( Actinobacteria Proteobacteria ( Proteobacteria It includes various phyla such as ) and others.
[0063] (3) At the genus level, the Lactobacillus genus in the MC group has probiotic characteristics ( Lactobacillus The relative abundance of [a specific organism] was significantly lower in the [a specific organism] group than in the NC group, while in the HP group, its abundance recovered to near normal levels. KLDS1.0344 intervention also altered the abundance of the [a specific organism] family NK4A136 group ([a specific organism]). Lachnospiraceae_NK4A136_group Bacteroides ( Muribaculum ), Clostridium genus ( Lachnoclostridium ), Riken Mycology ( Rikenellaceae The relative abundance of several core bacterial genera, such as )
[0064] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.
[0065] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
[0066] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.
Claims
1. A strain of *Lactobacillus plantarum* KLDS1.0344, its taxonomic name is... Lactobacillus plantarum It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 2022803.
2. A bacterial suspension, characterized in that, Containing *Lactobacillus plantarum* KLDS1.0344 as described in claim 1, and wherein the viable bacteria concentration in the bacterial suspension is 1 × 10⁻⁶. 7 CFU / mL to 1×10 9 CFU / mL.
3. The method for preparing the *Lactobacillus plantarum* KLDS1.0344 bacterial suspension according to claim 2, characterized in that, Includes the following steps: S1: Lactobacillus plantarum KLDS1.0344 with accession number CCTCC NO: M 2022803 was inoculated into MRS liquid medium for activation culture; S2: Subculture the activated bacterial solution; S3: Collect the bacterial cells, wash them with sterile physiological saline and resuspend them to obtain the bacterial suspension.
4. A microbial composition, characterized in that, It contains *Lactobacillus plantarum* KLDS1.0344 as described in claim 1, or its bacterial cells, or the bacterial suspension as described in claim 2 as an active ingredient.
5. The microbial composition according to claim 4, characterized in that, The dosage form of the composition is food, health food, or medicine.
6. Use of the Lactobacillus plantarum KLDS1.0344 of claim 1, the bacterial suspension of claim 2, or the microbial composition of claim 4 in the preparation of articles for improving depressive symptoms or alleviating depressive mood.
7. The use according to claim 6, characterized in that, The product in question is an antidepressant.
8. The use according to claim 6 or 7, characterized in that, The improvement of depressive symptoms or relief of depressive mood includes at least one of the following: improving behavioral hopelessness, alleviating anhedonia, and enhancing the ability to explore activities.
9. The use according to claim 6 or 7, characterized in that, The product exerts its effects through at least one of the following pathways: regulating the function of the hypothalamus-pituitary-adrenal axis, regulating the level of monoamine neurotransmitters, and increasing the level of brain-derived neurotrophic factor.
10. The microbial composition according to claim 4, characterized in that, The composition can be used as an effective ingredient in the preparation of various dosage forms, and can work together with other functional ingredients to prepare food, health food or medicine.