Use of lactobacillus plantarum or a preparation thereof in the production of uric acid in dogs, in the preparation of products that inhibit binge eating and / or promote self-execution

Abstract

This invention discloses the application of *Lactobacillus plantarum* or its products in the production of kynurenic acid, the preparation of products that inhibit overeating and / or promote spontaneous behavior, and belongs to the field of microbial technology. This *Lactobacillus plantarum* (… Lactiplantibacillus plantarum NHNK-2101 can synthesize and promote the production of kynurenic acid by intestinal epithelial cells, targeting and regulating the brain-gut axis function, reducing dietary preferences for high-sugar and high-fat foods, and improving spontaneous executive function. This invention also provides a specific method for producing kynurenic acid by culturing this strain or by co-culturing it with intestinal epithelial cells. Compared to the high safety risks and standardization difficulties of fecal microbiota transplantation in existing technologies, this invention utilizes a single strain with clearly defined components and controllable safety to achieve safe and precise intervention in overeating behavior and its associated cognitive impairment, providing a novel solution for developing related microecological preparations or functional products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of *Lactobacillus plantarum* or its products in the production of kyridine, the preparation of products that inhibit overeating and / or promote spontaneous behavior. Background Technology

[0002] Binge eating disorder (BED) is a psychosocial disorder characterized by uncontrolled, rapid, and excessive eating, often manifested by a strong craving for high-calorie, highly palatable, and ultra-processed foods. Long-term, recurrent episodes not only cause physiological damage but also lead to lasting changes in brain function, impairing core executive functions such as working memory and inhibitory control. Research indicates that gut microbiota dysbiosis plays a crucial role in this process. In particular, decreased levels of the gut microbiota metabolite kynurenic acid (KYNA) have been shown to overactivate the gut-brain axis neural circuits, thereby increasing food cravings, weakening behavioral control, and creating a vicious cycle of "binge eating leading to gut microbiota dysbiosis – dysbiosis exacerbating binge eating."

[0003] Currently, fecal microbiota transplantation (FMT) is the most representative intervention aimed at correcting gut microbiota dysbiosis. While it has shown potential in animal models to increase uric acid levels in dogs and improve overeating behavior, this technology faces significant challenges in clinical translation. These challenges primarily stem from: the complex preparation process, which carries safety risks such as pathogen transmission and immune rejection; and the difficulty in ensuring the stability and reproducibility of its therapeutic effects due to the complex and undefined composition of the transplanted material, making standardized production and precise regulation even more challenging. Furthermore, existing research largely focuses on suppressing the eating behavior itself, neglecting the deeper need to address the deeper issue of impaired spontaneous executive functioning caused by overeating through a safe and precise gut microbiota regulation approach, while simultaneously improving the behavior. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of *Lactobacillus plantarum* or its products in the production of kynurenic acid, the preparation of products that inhibit overeating and / or promote spontaneous behavior.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the invention provides the use of *Lactobacillus plantarum* or its products, said use comprising any one of the following: A1) Application in the production of canine uric acid; A2) Application in the preparation of products that inhibit overeating and / or promote spontaneous performance; The preservation number of the plant lactobacillus is CCTCC NO: M 2024802; The active ingredient in the product that inhibits overeating and / or promotes spontaneous behavior is *Lactobacillus plantarum*, *Lactobacillus plantarum* products, or kyridine.

[0006] Preferably, the suppression of overeating includes reducing the intake of high-sugar and high-fat foods and lowering fasting blood glucose.

[0007] Preferably, promoting spontaneous execution includes improving the ability to perform spontaneous execution.

[0008] Preferably, the *Lactobacillus plantarum* product is any one or more of live bacteria, inactivated bacteria, and fermentation products of *Lactobacillus plantarum*.

[0009] Preferably, the method for preparing the live bacteria includes: inoculating *Lactobacillus plantarum* into a culture medium, allowing it to stand and culture, obtaining a fermentation broth, centrifuging, taking the precipitate, washing it, and then obtaining live bacteria; The culture medium is MRS medium or MRS medium containing 0.2% (w / v) tryptophan.

[0010] Preferably, the method for preparing the fermentation product includes: inoculating *Lactobacillus plantarum* into a culture medium, allowing it to stand and culture, obtaining a fermentation broth, centrifuging, removing the supernatant, filtering, and obtaining the filtrate as the fermentation product; The culture medium is MRS medium containing 0.2% (w / v) tryptophan.

[0011] Preferably, the application in the production of canine uric acid includes static culture of *Lactobacillus plantarum*, followed by separation to obtain canine uric acid; The static culture medium used was MRS medium containing 0.2% (w / v) tryptophan.

[0012] Preferably, the application in the production of canine uric acid includes treating intestinal epithelial cells with *Lactobacillus plantarum* or its products, separating, and obtaining canine uric acid; The process includes culturing intestinal epithelial cells until they adhere to the culture medium, removing the cell culture medium, washing, adding DMEM medium and Lactobacillus plantarum or its products, and continuing to culture.

[0013] A second aspect of the present invention provides a method for producing kynurenic acid using *Lactobacillus plantarum* or its products, comprising: statically culturing *Lactobacillus plantarum*, separating, and obtaining kynurenic acid; The static culture medium used was MRS medium containing 0.2% (w / v) tryptophan; The static culture conditions are anaerobic static culture at 35-38℃ for 20-25 h; The preservation number of the *Lactobacillus plantarum* is CCTCC NO: M 2024802.

[0014] A third aspect of the present invention provides a method for producing kynurenic acid using *Lactobacillus plantarum* or its products, comprising: treating intestinal epithelial cells with *Lactobacillus plantarum* or its products, separating, and obtaining kynurenic acid; The process includes culturing intestinal epithelial cells until the cells adhere, removing the cell culture medium, washing, adding DMEM culture medium and Lactobacillus plantarum or its products, and culturing. The preservation number of the *Lactobacillus plantarum* is CCTCC NO: M 2024802.

[0015] The beneficial effects of this invention are as follows: This invention creatively applies the known *Lactobacillus plantarum* (CCTCC NO: M 2024802) to enhance kynurenic acid levels to improve binge eating and related behavioral disorders, achieving the following beneficial effects: Firstly, it reveals the novel application value of this strain in regulating the gut-brain axis, effectively producing and promoting intestinal kynurenic acid production. This provides a standardized, safe, and controllable treatment for binge eating caused by kynurenic acid deficiency, fundamentally overcoming the safety and standardization bottlenecks of fecal microbiota transplantation. Secondly, this application unexpectedly achieves synergistic intervention on binge eating behavior and its core cognitive impairment, simultaneously improving spontaneous executive function related to executive function while reducing preferences for high-sugar and high-fat foods. Furthermore, the application based on a single, clearly defined strain lays a reliable foundation for developing stable, industrially viable microecological preparations or functional products, significantly promoting the feasibility of clinical translation in this field.

[0016] Biological Preservation Instructions: The *Lactobacillus plantarum* described in this invention is *Lactobacillus plantarum* with the preservation number CCTCC NO: M 2024802. Lactiplantibacillus plantarum NHNK-2101 was deposited on April 25, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. This bacterium has been disclosed in a patent (application number: 2024107908001, invention title: A strain of Lactobacillus plantarum and its application in the preparation of products that reduce high levels of bilirubin). Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1 This is the device for applying foot electric shocks to mice in Embodiment 3 of the present invention.

[0019] Figure 2 Examples of normal and high-sugar, high-fat diets for mice in Example 3 of this invention are shown in the diagram.

[0020] Figure 3 This is a graph showing the results of the regulation of dietary preferences in mice by *Lactobacillus plantarum* NHNK-2101 in Example 3 of the present invention.

[0021] Figure 4 The graph shows the results of regulating fasting blood glucose in mice using *Lactobacillus plantarum* NHNK-2101 in Example 3 of this invention.

[0022] Figure 5 This is an example diagram illustrating the regulation of nest-building behavior in mice by *Lactobacillus plantarum* NHNK-2101 in Example 3 of the present invention.

[0023] Figure 6 This is a graph showing the nesting behavior scoring results of mice in Example 3 of the present invention. Detailed Implementation

[0024] Terminology Explanation: In this invention, kynurenic acid (KYNA) is a heterocyclic organic acid formed by the deamination and activation of kynuronic acid protons during tryptophan catabolism, and can be produced by intestinal flora metabolism. In mouse models of bulimia and human patients, the level of kynurenic acid in the intestines is significantly reduced. Studies have shown that the decrease in kynurenic acid levels is closely related to an excessive preference for high-calorie, flavorful foods, and binge eating behavior. When kynurenic acid levels are insufficient, the enterovagus nerve-nucleus solitarius-paraventricular nucleus circuit is overexcited, leading to increased food cravings in the brain and weakened control over eating, thus triggering bulimia.

[0025] In this invention, spontaneous executive functioning refers to an individual's ability to autonomously initiate, plan, and execute goal-oriented behaviors without external instructions, cues, or a structured environment. Binge eating can affect cognitive function, and executive function is one of the most important dimensions of cognitive symptoms, including working memory, higher-order cognitive abilities, inhibitory control, cognitive flexibility, and the ability to plan, reason, and solve problems. Current research on spontaneous executive function often assesses its strength by observing certain spontaneous behaviors in animals (such as nesting experiments in mice). Attention deficit hyperactivity disorder (ADHD) and frontotemporal dementia can both potentially lead to a lack of spontaneity.

[0026] Existing technologies that use fecal microbiota transplantation (FMT) to correct gut microbiota and improve binge eating behavior suffer from low safety, difficulty in standardization, and challenges in simultaneously repairing related cognitive impairments. To address these issues, this invention proposes a technical solution using *Lactobacillus plantarum* (CCTCC NO: M 2024802) or its products for kynurenic acid production, and further using it to prepare products that inhibit binge eating and promote spontaneous executive function. This solution leverages the key characteristic of this strain—its ability to produce and promote kynurenic acid production in the gut—to precisely intervene in the pathological aspects of binge eating at the source of the gut-brain axis. This not only avoids the safety risks of FMT and provides a foundation for developing standardized formulations, but also unexpectedly achieves the dual effects of inhibiting binge eating behavior and improving executive function, thus providing a safer, more precise, and comprehensive new treatment strategy.

[0027] A first typical embodiment of the present invention provides the application of *Lactobacillus plantarum* or its products, the application including any one of the following; A1) Application in the production of canine uric acid; A2) Application in the preparation of products that inhibit overeating and / or promote spontaneous performance; The preservation number of the plant lactobacillus is CCTCC NO: M 2024802; The active ingredient in the product that inhibits overeating and / or promotes spontaneous behavior is *Lactobacillus plantarum*, *Lactobacillus plantarum* products, or kyridine.

[0028] The plant lactobacillus disclosed in this invention ( Lactiplantibacillus plantarum NHNK-2101 has the ability to produce kynurenic acid, promote the production of kynurenic acid by intestinal epithelial cells, reduce dietary preferences for high-sugar and high-fat foods, and improve spontaneous execution. It can be used for kynurenic acid production and can also be used to prepare products that inhibit overeating and promote spontaneous execution.

[0029] In this invention, the products include, but are not limited to, functional foods, health products, pharmaceuticals, medical foods, scientific research and raw material products, or pet health products.

[0030] Among these, functional foods and health foods are products focused on weight management, mood regulation, and cognitive enhancement. For example, probiotic supplements, which are live bacteria preparations in the form of freeze-dried powder, capsules, or tablets, primarily target the gut-brain axis for regulation and are used for daily mood management and appetite control. Functional solid beverages or yogurts, which add *Lactobacillus plantarum* or its products to fermented milk, beverages, or meal replacements, are positioned as weight management or mood health foods to suppress overeating.

[0031] Pharmaceuticals or medical products focus on interventions for pathological conditions such as bulimia nervosa and executive dysfunction. For example, there are drugs used to treat bulimia nervosa (BED), impulsive eating associated with obesity, or to improve executive function decline associated with diseases such as ADHD and depression. There are also nutritional intervention products designed for specific disease populations (such as patients with metabolic syndrome) that help control appetite and cognitive behavior by regulating the gut microbiota.

[0032] Research and raw material products can be produced by fermenting this strain to produce kynauric acid, which can be used as a standard, research reagent or raw material for downstream functional products; or they can be postbiotic preparations, which contain a mixture of inactivated bacteria and metabolites, for use in scenarios where people are sensitive to or cannot ingest live bacteria.

[0033] Given the effectiveness of this technology in animal model studies, it can be transferred in parallel to the pet industry, for example, to pet probiotics or food for improving anxious eating, obesity, or executive compliance during behavioral training.

[0034] In this invention, suppressing overeating includes reducing the intake of high-sugar and high-fat foods and lowering fasting blood glucose.

[0035] Targeting the core trigger of binge eating—high-sugar, high-fat foods—this strain or its products can effectively intervene in reward-based eating behavior, reducing cravings for unhealthy foods rather than indiscriminate anorexia. Furthermore, this strain or its products can lower fasting blood glucose levels, directly linked to improved insulin sensitivity. Therefore, this application not only controls immediate eating urges but also addresses the underlying glucose metabolism disorders often associated with binge eating.

[0036] In this invention, promoting spontaneous execution includes improving the ability to perform spontaneous execution.

[0037] Existing technologies (such as fecal microbiota transplantation) typically focus on suppressing binge eating behavior itself. However, this invention, by increasing kynurenic acid levels, not only reduces the intake of high-sugar, high-fat foods in model animals but also unexpectedly and synergistically improves their ability to initiate, plan, and execute goal-oriented behaviors as demonstrated in nesting experiments. This proves that the intervention of this invention can reach deeper neurocognitive deficits behind binge eating behavior, achieving a qualitative change from symptom control to functional improvement. Spontaneous executive function is mainly related to the function of the prefrontal cortex. This effect strongly demonstrates that regulating the gut microbiota through specific strains (increasing kynurenic acid) can remotely and effectively influence brain regions related to higher cognitive functions. This provides functional evidence for the role of gut-brain bidirectional communication in complex behavioral regulation, making the mechanistic chain of brain-gut axis regulation more complete. This effect allows the application of this invention (such as probiotic preparations) to not only control binge eating behavior itself but also to improve problems such as decreased executive function and weakened decision-making ability often associated with binge eaters, giving the product unique additional functional positioning such as improving emotional eating and enhancing self-management.

[0038] In this invention, the *Lactobacillus plantarum* product is any one or more of live bacteria, inactivated bacteria, and fermentation products of *Lactobacillus plantarum*.

[0039] In this invention, the method for preparing the live bacteria includes: inoculating *Lactobacillus plantarum* into a culture medium, allowing it to stand and culture, obtaining a fermentation broth, centrifuging, taking the precipitate, washing it, and then obtaining live bacteria. The culture medium is MRS medium or MRS medium containing 0.2% (w / v) tryptophan.

[0040] The static culture conditions are as follows: anaerobic static culture at 35-38℃ for 20-25 h, preferably at 37℃ for 24 h. After static culture, the fermentation broth is obtained, centrifuged at 5000 rpm for 10 min, the precipitate is collected, and the precipitate is washed to obtain live bacteria.

[0041] In this invention, live bacteria are inactivated to obtain inactivated bacterial cells. This invention does not limit the inactivation method; those skilled in the art can use conventional inactivation methods, such as inactivation at 120-125°C for 10-20 min, preferably at 121°C for 15 min.

[0042] In this invention, the method for preparing the fermentation product includes: inoculating *Lactobacillus plantarum* into a culture medium, allowing it to stand and culture, obtaining a fermentation broth, centrifuging, removing the supernatant, filtering, and obtaining the filtrate as the fermentation product; The culture medium is MRS medium containing 0.2% (w / v) tryptophan.

[0043] The static culture conditions are as follows: anaerobic static culture at 35-38℃ for 20-25 h, preferably at 37℃ for 24 h. After static culture, the fermentation broth is obtained, centrifuged at 5000 rpm for 10 min, and the supernatant is collected and filtered with a precision of 0.22 μm. The obtained filtrate is the fermentation product.

[0044] In this invention, the application in the production of canine uric acid includes static culture of *Lactobacillus plantarum*, separation, and obtaining canine uric acid. The static culture medium used is MRS medium containing 0.2% (w / v) tryptophan. The static culture conditions are anaerobic static culture at 35-38℃ for 20-25 h, preferably at 37℃ for 24 h.

[0045] In this invention, the application in the production of canine uric acid includes treating intestinal epithelial cells with Lactobacillus plantarum or its products, separating and obtaining canine uric acid; The process includes culturing intestinal epithelial cells until they adhere to the culture medium, removing the cell culture medium, washing, adding DMEM medium and Lactobacillus plantarum or its products, and culturing.

[0046] The separation described in this invention aims to define the source of kynauric acid (KYNA), rather than limiting a specific purification process. In the field of microbial fermentation, obtaining the target metabolite (kynauric acid) from the fermentation broth of *Lactobacillus plantarum* is a routine technical operation. Those skilled in the art can flexibly choose separation methods according to the actual application scenario (such as the preparation of drugs, functional foods, or standards), as long as kynauric acid can be effectively enriched or extracted in the end.

[0047] Conventional separation methods include, but are not limited to, centrifugation or filtration (to remove bacterial cells and insoluble matter), organic solvent extraction (such as ethyl acetate), membrane separation (ultrafiltration, nanofiltration), chromatography (such as ion exchange chromatography, reversed-phase chromatography), and crystallization purification. These methods are all well-known and mature technologies in the field, and their selection and combination do not constitute a substantial contribution to the invention itself.

[0048] A second typical embodiment of the present invention provides a method for producing kynurenic acid using *Lactobacillus plantarum* or its products, comprising: statically culturing *Lactobacillus plantarum*, separating, and obtaining kynurenic acid; The static culture medium used was MRS medium containing 0.2% (w / v) tryptophan; The static culture conditions are anaerobic static culture at 35-38℃ for 20-25 h; The preservation number of the *Lactobacillus plantarum* is CCTCC NO: M 2024802.

[0049] This invention achieves efficient and stable production of the target active substance kynurenic acid using a single, well-defined, and safe strain through fermentation under specific conditions (anaerobic static culture at 35-38℃ for 20-25 h) in MRS medium containing 0.2% (w / v) tryptophan. This process fundamentally overcomes the inherent drawbacks of fecal microbiota transplantation (FMT), such as complex composition, large batch-to-batch variability, and uncontrollable safety issues. It transforms the source of kynurenic acid from the complex and uncontrollable human microbiota into a standardized, industrially producible microbial fermentation process, thus providing a direct raw material production route for developing formulations with well-defined components, stable quality, and reliable safety (such as pharmaceuticals, probiotics, or functional foods).

[0050] Similarly, the separation described herein aims to define the source of kynauric acid (KYNA), rather than limiting a specific purification process. In the field of microbial fermentation, obtaining the target metabolite (kynauric acid) from the fermentation broth of *Lactobacillus plantarum* is a routine technical operation. Those skilled in the art can flexibly choose separation methods according to the actual application scenario (such as the preparation of drugs, functional foods, or standards), as long as kynauric acid can be effectively enriched or extracted in the end.

[0051] A third typical embodiment of the present invention provides a method for producing kynurenic acid using *Lactobacillus plantarum* or its products, comprising: treating intestinal epithelial cells with *Lactobacillus plantarum* or its products, separating, and obtaining kynurenic acid; The process includes culturing intestinal epithelial cells until the cells adhere, removing the cell culture medium, washing, adding DMEM culture medium and Lactobacillus plantarum or its products, and culturing. The preservation number of the *Lactobacillus plantarum* is CCTCC NO: M 2024802.

[0052] This invention utilizes *Lactobacillus plantarum* or its products to directly treat intestinal epithelial cells, demonstrating that this strain not only synthesizes kynurenic acid itself but also effectively promotes kynurenic acid production in intestinal host cells. This replicates and reinforces, in vitro, the key mechanism by which the strain may function in vivo—namely, increasing local kynurenic acid concentration in the intestine through gut-bacterium interaction. This provides solid in vitro experimental evidence for the application of *Lactobacillus plantarum* or its products, directly revealing an important mode of function for this strain (promoting host production), rather than solely relying on its own synthesis. This invention demonstrates at the cellular level that this strain can target the intestinal interface, providing a clear research direction and efficacy basis for the subsequent development of live bacterial preparations or postbiotic products based on this strain aimed at enhancing intestinal function.

[0053] Similarly, the separation described herein aims to define the source of kynauric acid (KYNA), rather than limiting a specific purification process. In the field of microbial fermentation, obtaining the target metabolite (kynauric acid) from the fermentation broth of *Lactobacillus plantarum* is a routine technical operation. Those skilled in the art can flexibly choose separation methods according to the actual application scenario (such as the preparation of drugs, functional foods, or standards), as long as kynauric acid can be effectively enriched or extracted in the end.

[0054] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0055] In the following examples, the MRS solid and liquid culture media and related reagents used were all from Qingdao Haibo Biotechnology Co., Ltd., the DMEM culture medium was from Beijing Solarbio Technology Co., Ltd., the fetal bovine serum (FBS) was from Biological Industries (BI), and the sterile PBS used was from Hefei Baisha Biotechnology Co., Ltd. (0.01 M, pH=7.2).

[0056] Unless otherwise specified, the reagents and consumables used in this invention are all commercially available products.

[0057] Example 1: NHNK-2101 produces kynurenic acid 1. Preparation of NHNK-2101 fermentation products Single colonies of *Lactobacillus plantarum* NHNK-2101 were picked and cultured in MRS liquid medium containing 0.2% (w / v) tryptophan. After anaerobic static culture at 37°C for 24 h, the culture medium was adjusted to OD using MRS liquid medium containing 0.2% (w / v) tryptophan. 600 Centrifuge at 0.5, 5000 rpm for 10 min, collect the supernatant, and then filter through a 0.22 μm filter membrane to obtain sterile fermentation product.

[0058] 2. Establishment of the kynurenic acid standard curve A standard curve for kynurenine (KYN) was prepared using a kynurenine ELISA research kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.). The assay method was performed according to the kit instructions. 50 μL of each concentration of standard from the kit was added to an ELISA plate for the reaction. The absorbance at 450 nm was measured, and a standard curve was plotted.

[0059] The standard curve formula for kynurenic acid is: y = (A - D) / [1 + (x / C)^B] + D, where: A = 3.02150; B = 1.64778; C = 3.72691; D = 0.18235; R 2=0.99956, indicating good linearity and reliable method.

[0060] 3. Determination of kynurenic acid content produced by NHNK-2101 metabolism In the experimental group, 50 μL of sterile NHNK-2101 fermentation product was added to an ELISA plate for reaction. In the control group, an equal volume of MRS liquid medium containing 0.2% (w / v) tryptophan was added. The absorbance at 450 nm was measured and substituted into the standard curve to calculate the relative increase rate of kynurenic acid. The calculation formula was: Relative increase rate (%) = (Kynuretic acid concentration in experimental group / Kynurenic acid concentration in control group) × 100%. The results are shown in Table 1.

[0061] Table 1. Statistics on kynurenic acid produced by NHNK-2101 metabolism

[0062] Table 1 shows that *Lactobacillus plantarum* NHNK-2101 can metabolize tryptophan to produce kynurenic acid, with a relative increase rate of 132.60%–133.10%.

[0063] Example 2: NHNK-2101 promotes the production of kynurenic acid by intestinal epithelial cells 1. Preparation of live NHNK-2101 bacteria Single colonies of *Lactobacillus plantarum* NHNK-2101 were picked and cultured in MRS liquid medium at 37°C for 24 h. After culture, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min and the OD was adjusted with DMEM medium. 600 =0.3 for backup.

[0064] 2. Preparation of inactivated NHNK-2101 bacterial cells Single colonies of *Lactobacillus plantarum* NHNK-2101 were picked and cultured in MRS liquid medium at 37°C for 24 h. After culture, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min, autoclaved at 121°C for 15 min, and then the OD was adjusted with DMEM medium. 600 =0.3 for backup.

[0065] 3. Culture of Caco-2 intestinal epithelial cells Caco-2 cells were activated in DMEM medium containing 10% FBS and 1% penicillin and streptomycin, and then cultured at 37°C and 5% CO2. After the cells reached 80% to 90% confluence, they were passaged or plated.

[0066] 4. NHNK-2101-induced kynurenic acid secretion experiment from intestinal epithelial cells Caco-2 cells were divided into 1×10 6Cells were seeded per well in 6-well cell culture plates and cultured for 12 h until adherence. The culture medium was removed, and the cells were washed twice with sterile PBS. 1.9 mL of DMEM medium and 100 μL of live or inactivated NHNK-2101 bacteria were added to each well, while the control group received an equal volume of DMEM medium. The cells were incubated at 37°C for 24 h. After incubation, the supernatant was collected, and the kynurenic acid content was determined using a kynurenic acid (KYN) ELISA research kit.

[0067] Calculation formula: Relative increase rate (%) = 100% × experimental group OD 450 / Control group OD 450 The results are shown in Table 2.

[0068] Table 2. NHNK-2101 promotes the secretion of kynuric acid by Caco-2 cells.

[0069] Table 2 shows that both live and inactivated NHNK-2101 cells promoted the secretion of kynuric acid by Caco-2 cells, with a relative increase rate of 115.59%–149.35%. The fact that inactivated NHNK-2101 cells could promote kynuric acid secretion by Caco-2 cells also confirms that the increased kynuric acid did not solely originate from the metabolism of NHNK-2101 itself; the heat-stable active substances on inactivated NHNK-2101 cells could promote kynuric acid secretion by Caco-2 cells.

[0070] Example 3: NHNK-2101 reduces binge eating behavior and promotes spontaneous executive function. 1. Preparation of NHNK-2101 live bacterial suspension Single colonies of *Lactobacillus plantarum* NHNK-2101 were picked and cultured in MRS liquid medium at 37°C for 24 h. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with sterile PBS, followed by resuspending in sterile PBS to adjust the bacterial count to 2.7 × 10⁻⁶. 9 The cfu / mL concentration yields the NHNK-2101 live bacterial suspension.

[0071] 2. Mouse experimental grouping and treatment Twenty-four 6-week-old SPF (specific pathogen free) male C57BL / 6 mice were randomly divided into three groups of eight mice each: a control group, a model group, and an NHNK-2101 group. All mice were housed in an SPF-grade barrier enclosure, individually in each cage, with free access to food and water.

[0072] The experiment lasted 40 days. From day 0 to 7, mice were administered antibiotics via gavage to disrupt the pre-existing gut microbiota. Ampicillin 15 mg / kg, metronidazole 15 mg / kg, neomycin 25 mg / kg, and vancomycin 12.5 mg / kg were mixed and dissolved in sterile water. The model group and NHNK-2101 group received 10 mL / kg of antibiotics via gavage, while the control group received the same amount of sterile water via gavage. This was repeated for 7 consecutive days. From day 8 to 40, three cycles of diet-stress were implemented to induce a binge-eating behavior model in mice. The control group received free access to normal feed daily from day 8 to 40. The model group and NHNK-2101 group underwent diet-stress cycle induction as follows: 8-11 days: Fasting period, each mouse was given 1 g of normal feed per day.

[0073] 12-13 days: Induction period for binge eating behavior, with free access to normal feed plus high-sugar, high-fat feed (Oreo biscuits).

[0074] 14-17 days: Recovery period, free access to normal feed.

[0075] Day 18: Test day, acute stress was applied (15 foot shocks, 0.45 mA, 500 ms / shock).

[0076] The second and third cycles begin at 19 days and 30 days, respectively. The device for applying foot shocks to the mice is as follows: Figure 1 .

[0077] Starting from day 8, the NHNK-2101 group was administered 0.1 mL of NHNK-2101 live bacterial suspension by gavage for 33 days, while the model group and control group were administered an equal volume of PBS solution by gavage. On days 18, 29, and 40, the intake of high-sugar, high-fat diet (Oreo cookies) and normal diet per mouse was measured 24 hours after food storage. The ratio of normal diet to high-sugar, high-fat diet weight per mouse represents the mouse's dietary preference; a lower ratio indicates a stronger preference for the high-sugar, high-fat diet. The analysis results are as follows: Figure 2 . Figure 2 The results showed that the ratio of normal diet intake to high-sugar, high-fat diet (Oreo cookies) intake in the model group mice gradually decreased as the binge-feeding cycle progressed, indicating that the mice's preference for high-sugar, high-fat diet became increasingly pronounced. Intervention with NHNK-2101 increased the ratio of normal diet intake to high-sugar, high-fat diet intake in mice, thereby reducing their preference for high-sugar, high-fat diet.

[0078] Figure 3 These are example photos of mice at 40 days old, showing the remaining normal diet and the high-sugar, high-fat diet (Oreo cookies). Figure 3The results showed that the mice in the control group had higher levels of uneaten food from both types of diet, indicating that they did not have a strong preference for high-sugar, high-fat diets under normal conditions, providing a baseline for the abnormal preference in the model group. The model group almost completely consumed the Oreo cookies, demonstrating a significant preference for high-sugar, high-fat diets. The NHNK-2101 group had significantly higher levels of uneaten Oreo cookies than the model group, and also more uneaten normal food, proving that NHNK-2101 intervention can increase the intake ratio of normal diet to high-sugar, high-fat diets and reduce the preference for high-sugar, high-fat diets.

[0079] On days 18, 29, and 40, mice were restrained in a specialized restraint device with their tails fully exposed. The tails were disinfected by repeatedly wiping them with 75% alcohol swabs. The tip of the tail was then trimmed by 1-2 mm using sterile scissors. The tail was gently massaged from the base to the tip, and venous blood was collected from the tail. Fasting blood glucose levels were measured, and the results were analyzed as follows: Figure 4 As shown.

[0080] Figure 4 The results showed that fasting blood glucose levels in the control group mice remained relatively stable, while those in the model group mice significantly increased during the test period, indicating that a preference for a high-sugar, high-fat diet further increased fasting blood glucose levels. NHNK-2101 further reduced fasting blood glucose levels in mice by decreasing their preference for a high-sugar, high-fat diet. During the test period, fasting blood glucose levels in the NHNK-2101 group mice remained relatively stable. This further confirms that intervention with NHNK-2101 can reduce mice's preference for a high-sugar, high-fat diet and improve the increase in fasting blood glucose caused by dietary preferences.

[0081] On day 37, a nesting experiment was conducted on mice. Corn cob bedding approximately 1 cm thick was introduced into the cages, and 10 g of uniformly cut rectangular (4 cm × 2 cm) thin cotton sheets were evenly placed in the cages 2 hours before the start of the nighttime circadian rhythm. The nesting quality of the mice was scored 18 hours later. The scoring criteria are as follows: 1 point; the scraps of paper were scattered haphazardly throughout the cage, but had not been torn or bitten. 2 points. The paper pieces were gathered to one side of the cage, but were loose and did not form a nest. There was no obvious tearing or folding. 3 points, the paper pieces are gathered and folded into a shaped but relatively flat nest, without obvious tearing or biting; 4 points. The paper pieces are gathered and folded into a deep nest and torn into small pieces.

[0082] All mice were scored using a blinded method. See the attached images for examples of nesting behavior in different groups. Figure 5 The results of Lactobacillus plantarum NHNK-2101 regulating nesting behavior in mice (nesting score) are shown in the figure. Figure 6 As shown.

[0083] Figure 5 The results showed that the nests built by mice in the blank control group were more complete, while the nests built by mice in the model group were irregular and less complete. The nest-building behavior of mice in the NHNK-2101 group was restored and the nests were more complete. Figure 6 The results showed that, compared with the control group, the nesting behavior score of mice in the model group was reduced, while *Lactobacillus plantarum* NHNK-2101 increased the nesting behavior score of mice. Nesting behavior is a comprehensive, non-invasive behavioral indicator for assessing spontaneous executive function in mice. Therefore, by using the nesting behavior score of mice, it can be determined that *Lactobacillus plantarum* NHNK-2101 has the effect of improving spontaneous executive function in mice.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of *Lactobacillus plantarum* or its products, characterized in that, The application includes any one of the following: A1) Application in the production of canine uric acid; A2) Application in the preparation of products that inhibit overeating and / or promote spontaneous performance; The preservation number of the plant lactobacillus is CCTCC NO: M 2024802; The active ingredient in the product that inhibits overeating and / or promotes spontaneous behavior is *Lactobacillus plantarum*, *Lactobacillus plantarum* products, or kyridine.

2. The application as described in claim 1, characterized in that, Suppressing overeating includes reducing the intake of high-sugar and high-fat foods and lowering fasting blood glucose.

3. The application as described in claim 1, characterized in that, The promotion of spontaneous execution includes improving the capacity for spontaneous execution.

4. The application as described in claim 1, characterized in that, The Lactobacillus plantarum product is any one or more of the following: live Lactobacillus plantarum cells, inactivated cells, and fermentation products.

5. The application as described in claim 4, characterized in that, The method for preparing the live bacteria includes: inoculating *Lactobacillus plantarum* into a culture medium, allowing it to be cultured statically to obtain a fermentation broth, centrifuging, taking the precipitate, washing it, and then obtaining the live bacteria. The culture medium is MRS medium or MRS medium containing 0.2% (w / v) tryptophan.

6. The application as described in claim 4, characterized in that, The method for preparing the fermentation product includes: inoculating *Lactobacillus plantarum* into a culture medium, allowing it to be cultured statically to obtain a fermentation broth, centrifuging, removing the supernatant, filtering, and obtaining the filtrate as the fermentation product; The culture medium is MRS medium containing 0.2% (w / v) tryptophan.

7. The application as described in claim 1, characterized in that, The application in the production of canine uric acid includes static culture of *Lactobacillus plantarum*, followed by separation to obtain canine uric acid. The static culture medium used was MRS medium containing 0.2% (w / v) tryptophan.

8. The application as described in claim 1, characterized in that, The application in the production of canine uric acid includes treating intestinal epithelial cells with Lactobacillus plantarum or its products, separating and obtaining canine uric acid; The process includes culturing intestinal epithelial cells until they adhere to the culture medium, removing the cell culture medium, washing, adding DMEM medium and Lactobacillus plantarum or its products, and culturing.

9. A method for producing canine uric acid using *Lactobacillus plantarum* or its products, characterized in that, include: Keninuric acid was obtained by static culture and isolation of *Lactobacillus plantarum*. The static culture medium used was MRS medium containing 0.2% (w / v) tryptophan; The static culture conditions are anaerobic static culture at 35-38℃ for 20-25 h; The preservation number of the *Lactobacillus plantarum* is CCTCC NO: M 2024802.

10. A method for producing canine uric acid using *Lactobacillus plantarum* or its products, characterized in that, include: Intestinal epithelial cells were treated with Lactobacillus plantarum or its products, and then kynurenic acid was isolated and obtained. The process includes culturing intestinal epithelial cells until the cells adhere, removing the cell culture medium, washing, adding DMEM culture medium and Lactobacillus plantarum or its products, and continuing to culture. The preservation number of the *Lactobacillus plantarum* is CCTCC NO: M 2024802.

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