Application of lactobacillus rhamnosus LG-1 metaplasm in relieving female depression

Abstract

The invention discloses an application of a lactobacillus rhamnosus LG-1 postbiotic in relieving female depression, the lactobacillus rhamnosus LG-1 postbiotic is one or more of a lactobacillus rhamnosus LG-1 live strain, an inactivated strain and a strain metabolite, and the live bacterium concentration of the lactobacillus rhamnosus LG-1 postbiotic is greater than or equal to 1 * 10 < 9 > CFU. The lactobacillus rhamnosus LG-1 disclosed by the invention shows a remarkable effect in the aspect of treating female depression, and the lactobacillus rhamnosus LG-1 can reach gastrointestinal mucosa through a strong acid environment in the stomach so as to regulate and optimize an intestinal probiotic composition. The micromolecular substances contained in the thallus metabolite can regulate the mood of a host through an intestine-brain axis, can provide a nutritional environment for intestinal probiotics, and is beneficial to proliferation of normal intestinal flora, so that the intestinal micro-ecological flora tends to be balanced. And a biogen product prepared by compounding the two can be used for micro-ecological treatment of depression.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of Lactobacillus rhamnosus LG-1 postbiotic in alleviating female depression. Background Technology

[0002] Depression is a common mental illness, affecting approximately 300 million people worldwide. According to the latest epidemiological study of adult depressive disorders in my country, the lifetime prevalence of depressive disorders is 6.8%. However, among patients with depressive disorders, only 9.5% have received treatment, and only 0.5% have received adequate treatment. Depression severely impacts patients' quality of life, reduces their learning and work efficiency, increases the burden on patients and their families, and can even lead to suicide, causing serious harm to patients, families, and society. First-line treatment for depression primarily involves drugs that correct central monoamine neurotransmitter imbalances, such as serotonin reuptake inhibitors (STIs) and dual STI / norepinephrine reuptake inhibitors. However, these drugs have drawbacks such as low response rates and significant side effects, mainly manifested in: ① slow clinical onset, with a delayed effect; generally, it takes 2-3 weeks for clinical efficacy to appear after taking antidepressants. ② low treatment response rate; only one-third of patients with depression respond to initial antidepressant treatment. ③ significant drug resistance; treatment-resistant depression has become a major challenge in the treatment of depression. ④ Increases the suicide risk for patients with depression, especially adolescents. Clearly, drug treatment for depression faces significant challenges, and there is an urgent need to find new treatment options.

[0003] Significant gender differences exist in depression, with women exhibiting more risk factors and the prevalence of MDD (demand-induced depression) in women of reproductive age being more than twice that of men of the same age. Hormonal fluctuations are particularly pronounced during menstruation, pregnancy, childbirth, lactation, menopause, and perimenopause, making women more susceptible to depressive moods, such as premenstrual syndrome, prenatal / postpartum depression, and perimenopausal depression. While significant gender differences may exist in the pathogenesis and treatment of depression, there are currently no specific treatment protocols for women with depression. In most studies, gender is treated merely as an important biological variation factor, especially in animal experiments where female animals are often excluded. Some domestic and international studies have shown that sex hormone supplementation can enhance the antidepressant effect in female patients; however, the use of sex hormone preparations remains highly controversial. Therefore, there is an urgent need to develop safe and effective antidepressant products for women with depression.

[0004] Microbiome intervention or probiotic therapy holds promise as a new strategy for treating major diseases. However, current probiotic products on the market focus on supplementing gut microbiota without in-depth research into the specific benefits of strains to the host. In September 2018, Cell magazine published two articles simultaneously, concluding that "probiotics need to be personalized" and "probiotics are not conducive to gut microbiota recovery after antibiotic use," pushing probiotics into the spotlight and suggesting caution regarding the overheated probiotic market. Another systematic review included 32 randomized clinical trials evaluating the effects of probiotic preparations (including probiotics, prebiotics, and synbiotics) on adult depressive symptoms; only 7 studies showed significant antidepressant effects. The current more objective view is that universally applicable probiotics may not exist, and probiotics should not be used indiscriminately; personalized gut microbiota regulation strategies are the future trend.

[0005] Lactobacillus rhamnosus is a widely studied probiotic that can survive under harsh conditions in the digestive tract, including strong acid, bile, and the presence of various digestive enzymes, and can colonize the gastrointestinal mucosa to perform important physiological functions. Lactobacillus rhamnosus LGR-1 was first isolated from the female vagina and is widely considered beneficial to women's health; estrogen can upregulate the abundance of LGR-1 in the body's local microenvironment. In preliminary experiments of this invention, it was found that continuous administration of LGR-1 increased food and water intake in male mice and significantly improved their motor function compared to the control group. Female mice showed no significant changes in food and water intake or motor function. Female mice exhibited anti-anxiety and antidepressant effects, while male mice showed no significant anti-anxiety and antidepressant effects. This again emphasizes the importance of considering the suitability of probiotics and avoiding ineffective misuse.

[0006] Epigenetics are a collective term for the metabolites of probiotics after processing, including the bacterial cells and metabolites. Even after heat inactivation or treatment with gastrointestinal digestive fluids, heat-inactivated epigenetics retain high physiological activity. The rise of epigenetics represents the third revolution in the probiotic industry, and internationally, epigenetics are enabling lactic acid bacteria products to become more diverse and innovative. Small molecules secreted, modified, or degraded by bacterial flora can regulate host health. Therapies based on these metabolites are called epigenetic therapies. Epigenetic therapies do not target the bacterial flora itself, but rather target downstream pathways of the bacterial flora. By administering exogenous drugs or inhibiting metabolites, they restore the balance of bacterial metabolites, counteracting / correcting the effects of microecological imbalances. They have potential advantages such as low toxicity and dosage and administration routes that conform to pharmacokinetic principles. The screening of epigenetics has significant social and research translational value. Therefore, developing an epigenetic product to alleviate depression in women will provide more, safer, and more effective strategies for the microecological treatment of depression, and also has significant social implications and enormous market value. Summary of the Invention

[0007] Therefore, this invention provides the application of Lactobacillus rhamnosus LG-1 postbiotic in alleviating female depression.

[0008] The Lactobacillus rhamnosus LG-1 postbiotic provided by this invention is one or more of the following: live Lactobacillus rhamnosus LG-1 strain, inactivated strain, and strain metabolites.

[0009] The viable bacterial concentration of Lactobacillus rhamnosus LG-1 postbiotic provided by this invention is ≥1×10⁻⁶. 9 CFU.

[0010] The present invention also discloses a method for inactivating live strain of Lactobacillus rhamnosus LG-1, comprising the following steps: Ultrasonic cell lysis was performed in an ice bath at a power of 300W, with a 5-second operation followed by a 10-second pause, for a total of 20 minutes at 4°C, to obtain the ultrasonically lysed bacterial solution.

[0011] In addition, the inactivation method for live Lactobacillus rhamnosus LG-1 strain can also be the heat inactivation method, which includes the following steps: placing the bacterial suspension at 90°C and heating it in a water bath for 30 minutes to obtain a heat-inactivated bacterial solution.

[0012] The Lactobacillus rhamnosus LG-1 postbiotic provided by this invention can be used as a single ingredient or additive in the preparation of products for the treatment or prevention of female depression.

[0013] The products for treating or preventing female depression prepared from Lactobacillus rhamnosus LG-1 postbiotic provided by this invention include, but are not limited to, pharmaceuticals, nutritional products, dietary supplements, and health products.

[0014] The carrier forms of products for treating or preventing female depression prepared from Lactobacillus rhamnosus LG-1 postbiotic provided by the present invention include, but are not limited to, enzymes, kimchi, solid beverages, pills, tablets, and microcapsules.

[0015] The beneficial effects of the present invention are as follows: The present invention, through forced swimming behavior, tail suspension test, open field behavior test and elevated cross maze behavior test on mice, confirmed that Lactobacillus rhamnosus LG-1 postbiotic has antidepressant effect on female mice.

[0016] Lactobacillus rhamnosus LG-1 has shown significant effects in alleviating depression in female mice. This bacterium can reach the gastrointestinal mucosa through the highly acidic environment of the stomach, thereby regulating and optimizing the combination of intestinal probiotics. The small molecules contained in the bacterial metabolites can regulate host mood through the gut-brain axis and provide a nutrient environment for intestinal probiotics, which is conducive to the proliferation of normal intestinal flora and makes the intestinal microecological flora tend to be balanced. The bio-preservative products made by combining the two can be used for the microecological treatment of depression. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a bar chart showing the behavioral results of forced swimming. Figure 2 This is a bar chart of the results of the tail suspension experiment; Figure 3 This is a bar chart of the results from open field behavior analysis; Figure 4 This is a bar chart showing the behavioral results of the elevated cross maze. Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The Lactobacillus rhamnosus LG-1 postbiotic provided by this invention is one or more of the following: live Lactobacillus rhamnosus LG-1 strain, inactivated strain, and strain metabolites.

[0021] The viable bacterial concentration of Lactobacillus rhamnosus LG-1 postbiotic provided by this invention is ≥1×10⁻⁶. 9 CFU.

[0022] The present invention also discloses a method for inactivating live strain of Lactobacillus rhamnosus LG-1, comprising the following steps: Ultrasonic cell lysis was performed under ice bath conditions at a power of 300W, with a 5-second operation followed by a 10-second pause, for a total of 20 minutes to obtain the ultrasonically lysed bacterial solution.

[0023] Methods for inactivating live strains of Lactobacillus rhamnosus LG-1 also include heat inactivation, which involves placing the bacterial suspension at 90°C and heating it in a water bath for 30 minutes to obtain a heat-inactivated bacterial solution.

[0024] The Lactobacillus rhamnosus LG-1 postbiotic provided by this invention can be used as a single ingredient or additive in the preparation of products for the treatment or prevention of female depression.

[0025] The products for treating or preventing female depression prepared from Lactobacillus rhamnosus LG-1 postbiotic provided by this invention include, but are not limited to, pharmaceuticals, nutritional products, dietary supplements, and health products.

[0026] The carrier forms of products for treating or preventing female depression prepared from Lactobacillus rhamnosus LG-1 postbiotic provided by the present invention include, but are not limited to, enzymes, kimchi, solid beverages, pills, tablets, and microcapsules.

[0027] The following experiment will further verify the efficacy of Lactobacillus rhamnosus LG-1 postbiotic in alleviating depression and anxiety in female mice: I. Main reagents: Laboratory animals: 6-8 week old female C57BL / 6 mice, averaging 18-20g each. All mice were housed in a specific pathogen-free (SPF) laboratory, with free access to food and water. The room temperature was maintained at 22±1℃, and the relative humidity was maintained at 55±5%. The diurnal cycle was set at 8:00 AM / 8:00 PM. Lactobacillus rhamnosus LG-1, ATCC55826, purchased from the American Type Culture Collection (ATCC). MRS liquid culture medium (1L): 10g peptone, 5g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 2g triammonium citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 1ml Tween 80.

[0028] MRS solid medium: Add 1.5-2% agar to the MRS liquid medium.

[0029] Before animal experiments, *Lactobacillus rhamnosus* LG-1 was activated by second-generation inoculation and then inoculated at 1% in MRS liquid medium. The culture was anaerobic at 37°C for 24 hours to obtain a bacterial suspension. The bacterial suspension was divided into three equal portions and processed as follows: (1) Live bacteria component (Active LG-1): The bacterial suspension was centrifuged at 10,000 rpm for 10 minutes at 4°C. After removing the supernatant, it was resuspended in fresh MRS culture medium and added to the drinking water of mice to adjust the final bacterial concentration to 1×10⁻⁶. 9 CFU; (2) Heat-killed bacterial solution (HK): The bacterial suspension was heated in a water bath at 90°C for 30 minutes to obtain a heat-killed bacterial solution. No viable bacteria grew when inoculated onto agar plates. The solution was added to the drinking water of mice, and the final concentration was adjusted according to the volume of the viable bacteria components mentioned above.

[0030] (3) Ultrasonic Lysates (UL): The bacterial suspension was subjected to ultrasonic cell lysis in an ice bath at a power of 300W, with a 5-second operation followed by a 10-second pause, for a total of 30 minutes. The suspension was then inoculated onto plates where no viable bacteria were observed to grow. The solution was added to the drinking water of mice, and the final concentration was adjusted according to the volume of the viable bacteria components described above.

[0031] (4) Bacterial metabolites (CFS, Cell-free Supernatant): The supernatant obtained by centrifuging the preparation of live bacteria components was filtered through a 0.22 μm filter membrane to obtain Lactobacillus rhamnosus LG-1 metabolites. No live bacteria grew when inoculated onto plates. The CFS was added to the drinking water of mice, and the final concentration was adjusted according to the volume of the live bacteria components mentioned above.

[0032] One week prior to the experiment, mice underwent acclimatization feeding and were randomly divided into 5 groups: a control group (Control, n=10), a live bacteria group (active LG-1, n=12), a heat-inactivated bacteria group (HK, n=12), an ultrasonically lysed bacterial culture group (UL, n=12), and a bacterial metabolite group (CFS, n=12). The prepared postbiotics were added to the drinking water of the corresponding mice at a final bacterial concentration of 1×10⁻⁶. 9 CFU concentration was adjusted. After 28 days of continuous administration, behavioral evaluations (forced swimming, tail suspension test, open field test, and elevated cross maze test) were conducted. The behavioral experiments were designed and performed mainly based on previous studies (Pan JX, et al. J Neurol Sci. 2019; 400:160-168; Zheng P, et al. Sci Adv. 2019; 5(2): eaau8317). All behavioral records were recorded using the ANY-maze animal behavior analysis system.

[0033] (1) Forced swimming: The test lasted for a total of 6 minutes. The first minute was the water adaptation phase. Starting from the second minute, the timer was 5 minutes. The number of times the mice in the two groups remained still in the water and the total duration were calculated within 5 minutes. The longer the total still time, the stronger the mice's sense of despair and helplessness, which means that the mice's depressive symptoms were more severe.

[0034] (2) Tail suspension test: Before each group of mice was tested, the inner wall of the tail suspension box was wiped with 75% alcohol. The tail of the mouse was fixed with tape, so that the mouse head was hanging down. The suspension point of the mouse tail was 50cm away from the experimental table. After acclimatization for 1 minute, the cumulative immobility time of the mouse in the last 5 minutes was recorded.

[0035] (3) Open field test: Mice were placed individually in a clean 44.5cm×44.5cm×45cm open field test chamber for 6 minutes. The activity of the mice in the open field was recorded in the last 5 minutes. The total distance of movement in the horizontal direction, the time spent in the central area, the percentage of movement distance, and the number of times the mice stood up in the vertical direction were analyzed. Before the experiment, the inner wall of the tail suspension box was wiped with 75% alcohol.

[0036] (4) Elevated Cross Maze Experiment: The elevated cross maze consists of two open arms and two closed arms (each arm is 5cm wide and 35cm long), intersecting in a cross shape. The intersection is the central area, which is 50cm above the ground. At the beginning of the experiment, mice are placed into the maze from the central grid towards the closed arms. The experiment lasts for 6 minutes. The number of times the mice enter the open arms, the time spent in the open arms, the number of times the mice enter the closed arms, and the time spent in the closed arms are recorded in the last 5 minutes. Before the experiment, the inner wall of the tail suspension box is wiped with 75% alcohol.

[0037] II. Test Results See Figure 1 The forced swimming behavioral results indicated that, compared with the control group, the immobility time of mice in the live bacteria group (active LG-1), heat-inactivated bacterial solution group (HK), ultrasonically lysed bacterial solution group (UL), and bacterial metabolite group (CFS) was significantly shorter than that of the control group. Control group: MRS group; Active LG-1 group: active Lactobacillus rhamnosus LG-1 group; HK group: heat-inactivated Lactobacillus rhamnosus LG-1 culture medium group; UL group: ultrasonically lysed Lactobacillus rhamnosus LG-1 culture medium group; CFS group: Lactobacillus rhamnosus LG-1 metabolite group. This suggests that live Lactobacillus rhamnosus, heat-inactivated bacterial solution, ultrasonically lysed bacterial solution, and metabolites have antidepressant effects on female mice.

[0038] See Figure 2 The tail suspension test results indicated that, compared with the control group, the immobility time of mice in the live bacteria group (active LG-1), heat-inactivated bacterial solution group (HK), ultrasonically lysed bacterial solution group (UL), and bacterial metabolite group (CFS) was significantly shorter than that in the control group. Control group: MRS group; Active LG-1 group: active Lactobacillus rhamnosus LG-1 group; HK group: heat-inactivated Lactobacillus rhamnosus LG-1 culture medium group; UL group: ultrasonically lysed Lactobacillus rhamnosus LG-1 culture medium group; CFS group: Lactobacillus rhamnosus LG-1 metabolite group. This suggests that live Lactobacillus rhamnosus, heat-inactivated bacterial solution, ultrasonically lysed bacterial solution, and metabolites have antidepressant effects on female mice.

[0039] See Figure 3Open field behavior results indicated that, compared with the control group, mice in the live bacteria group (active LG-1), heat-inactivated bacterial solution group (HK), ultrasonically lysed bacterial solution group (UL), and bacterial metabolite group (CFS) spent significantly more time in the central region. Control group: MRS group; Active LG-1 group: active Lactobacillus rhamnosus LG-1 group; HK group: heat-inactivated Lactobacillus rhamnosus LG-1 culture medium group; UL group: ultrasonically lysed Lactobacillus rhamnosus LG-1 culture medium group; CFS group: Lactobacillus rhamnosus LG-1 metabolite group. This suggests that live Lactobacillus rhamnosus, heat-inactivated bacterial solution, ultrasonically lysed bacterial solution, and metabolites have an anxiolytic effect on female mice.

[0040] See Figure 4 Behavioral results from the elevated cruciate maze indicated that, compared to the control group, the mice exerted significantly more effort at the open arm position. The control groups included: MRS group; Active LG-1 group (lactobacillus rhamnosus LG-1); HK group (heat-inactivated Lactobacillus rhamnosus LG-1 culture); UL group (ultrasonic lysis of Lactobacillus rhamnosus LG-1 culture); and CFS group (Lactobacillus rhamnosus LG-1 metabolites). This suggests that live Lactobacillus rhamnosus, heat-inactivated culture, ultrasonically lysed culture, and metabolites have an anxiolytic effect on female mice.

[0041] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

Claims

1. Application of Lactobacillus rhamnosus LG-1 postbiotic in alleviating female depression.

2. The application of Lactobacillus rhamnosus LG-1 postbiotic according to claim 1 in alleviating female depression, characterized in that, The Lactobacillus rhamnosus LG-1 metabiotic is one or more of the following: live Lactobacillus rhamnosus LG-1 strain, inactivated strain, and strain metabolites.

3. The application of *Lactobacillus rhamnosus* LG-1 postbiotic as described in claim 1 or 2 in alleviating female depression, characterized in that... The viable cell concentration of Lactobacillus rhamnosus LG-1 probiotic is ≥ 1 x 10 9 CFU.

4. The application of Lactobacillus rhamnosus LG-1 postbiotic according to claim 2 in alleviating female depression, characterized in that, The method for inactivating the live strain of Lactobacillus rhamnosus LG-1 includes the following steps: Ultrasonic cell lysis was performed under ice bath conditions at a power of 300W, with a 5-second operation followed by a 10-second pause, for a total of 20 minutes to obtain the ultrasonically lysed bacterial solution.

5. The application of Lactobacillus rhamnosus LG-1 postbiotic according to claim 2 in alleviating female depression, characterized in that, The method for inactivating the live strain of Lactobacillus rhamnosus LG-1 includes the following steps: placing the bacterial suspension at 90°C and heating it in a water bath for 30 minutes to obtain a heat-inactivated bacterial solution.

6. The application of Lactobacillus rhamnosus LG-1 postbiotic according to claim 3 in alleviating female depression, characterized in that, The Lactobacillus rhamnosus LG-1 postbiotic can be used as a single ingredient or additive in the preparation of products for the treatment or prevention of female depression.

7. The application of Lactobacillus rhamnosus LG-1 postbiotic according to claim 6 in alleviating female depression, characterized in that, Products prepared from Lactobacillus rhamnosus LG-1 postbiotics for the treatment or prevention of female depression include, but are not limited to, pharmaceuticals, nutritional products, dietary supplements, and health products.

8. The application of Lactobacillus rhamnosus LG-1 postbiotic according to claim 5 in alleviating female depression, characterized in that... Products for the treatment or prevention of depression in women prepared from Lactobacillus rhamnosus LG-1 postbiotics may be available in carrier forms including, but not limited to, enzymes, kimchi, solid beverages, pills, tablets, and microcapsules.

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