Novel Lactobacillus strains and their use

Abstract

This invention provides compositions and their uses for effective treatments to improve sleep quality. [Solution] The present invention provides a novel isolate of Lactobacillus gasseri, its culture product, and a method for producing the culture product of the isolate. The present invention also provides a method for improving sleep quality using the isolate or the culture product of the isolate.

Description

TECHNICAL FIELD

[0001] The present invention relates to a novel isolate of Lactobacillus gasseri, a culture product thereof, and a method for producing the culture product. The present invention also relates to a method for improving sleep quality using the isolate or the culture product thereof. BACKGROUND ART

[0002] Monoamine oxidase (MAO) is an important enzyme that mainly functions to degrade monoamine neurotransmitters including 5-hydroxytryptamine (5-HT), noradrenaline and dopamine. MAO is distributed in different brain regions, and has two main forms, MAO-A and MAO-B, which differ in substrate preference. MAO plays an important role in regulating the content of neurotransmitters, and thus indirectly affects mood, sleep and other physiological functions. MAO activity is associated with sleep quality: excessively high MAO activity causes the content of neurotransmitters (e.g., 5-HT) to become too low, thereby affecting sleep. A decrease in 5-HT content often leads to insomnia and reduced sleep quality. Studies have shown that, due to the lower neurotransmitter content in individuals with high MAO activity, such individuals are more likely to develop sleep disorders.

[0003] MAO inhibitors (MAOIs) improve mood and sleep by inhibiting the activity of MAO to increase the content of 5-HT, noradrenaline and dopamine. MAOIs are used for treating depression and certain sleep disorders, because they can improve sleep quality and mood states by increasing the bioavailability of these neurotransmitters.

[0004] 5-hydroxytryptamine (5-HT) is an important neurotransmitter widely present in the central nervous system, involved in regulating various physiological functions including mood, appetite, sexual activity, pain perception, and the sleep-wake cycle. A decrease in 5-HT may lead to insomnia and poor sleep quality. Studies have found that 5-HT transporter gene polymorphisms influence the development of obstructive sleep apnea syndrome.

[0005] Lactobacillus sp. is a common probiotic widely present in areas such as the intestinal tract, oral cavity, and reproductive tract, playing an important role in maintaining health and preventing disease. The main effects of Lactobacillus in the body include: (1) Improving intestinal health and maintaining the balance of the intestinal microecology: suppressing the growth of harmful bacteria (e.g., E. coli, Clostridioides difficile) and promoting the growth of beneficial bacteria. Promoting intestinal peristalsis: alleviating constipation and diarrhea, especially diarrhea associated with antibiotics. Strengthening the intestinal barrier function: helping to repair the intestinal mucosa and reducing leaky gut. (2) Immunomodulation: stimulating the activity of immune cells (e.g., macrophages, natural killer cells) and increasing the body's resistance to pathogens. Reducing inflammatory responses: reducing excessive immune responses and helping to control chronic inflammatory diseases such as allergies and enteritis. (3) Cholesterol reduction: Some Lactobacillus species can break down bile acids, reducing cholesterol absorption, which is beneficial for cardiovascular health. (4) Blood glucose stabilization: May help improve insulin sensitivity and aid in blood glucose control. (5) Anti-infective effects: Prevents urogenital tract infections, especially in women, and Lactobacillus plays an important role in maintaining the balance of the vaginal microecology, potentially reducing the risk of bacterial vaginosis and urinary tract infections. Suppression of oral pathogens: Helps reduce the number of oral pathogens, such as bacteria associated with plaque. (6) Improved skin health: Antioxidant, reducing damage from free radicals and helping to improve skin aging. Anti-inflammatory, and may have a soothing effect on sensitive skin or eczema. (7) Anti-cancer potential: Reduces the formation of carcinogens, and Lactobacillus can biodegrade several potential carcinogens. Enhanced immune surveillance: Helps the body's immune system identify and eliminate tumor cells. (8) Mental health support (gut-brain axis effect): Lactobacillus may improve mental health through the production of short-chain fatty acids (SCFAs) and their effect on neurotransmitters (e.g., serotonin), which may help alleviate symptoms of anxiety and depression.

[0006] Given the gradually increasing trend of declining sleep quality among modern people, effective treatments to improve sleep quality remain in demand. [Overview of the project]

[0007] In this invention, a novel strain of Lactobacillus gasseri has been discovered that can be used to improve sleep quality. Therefore, this invention provides the novel strain, related products, and methods for producing them.

[0008] In one embodiment, the Lactobacillus gasseri of the present invention has substantially the same characteristics as the strain with deposit number BCRC 911242 deposited with the Food Industry Development Research Institute. In a preferred embodiment, the strain contains the sequence SEQ ID NO: 1. In a more preferred embodiment, the strain is Lactobacillus gasseri LyLG1 with deposit number BCRC 911242 deposited with the Food Industry Development Research Institute.

[0009] The present invention also provides a method for producing a Lactobacillus gasseri culture product, comprising inoculating the Lactobacillus gasseri of the present invention into a suitable culture medium and culturing it under appropriate conditions to obtain the culture product.

[0010] In one embodiment, the culture conditions for Lactobacillus gasseri of the present invention are substantially the same as known methods for culturing Lactobacillus gasseri. In one embodiment, the culture conditions of the method can be adjusted as needed, for example, the culture medium components, culture temperature, culture time, etc. In a preferred embodiment, the culture medium is a solid medium or a liquid medium. In a more preferred embodiment, the culture medium is MRS medium.

[0011] The present invention also provides a culture product produced by Lactobacillus gasseri of the present invention. In one embodiment, the culture product can be obtained by a known method for culturing Lactobacillus. In a preferred embodiment, the culturing method includes culturing Lactobacillus gasseri until the logarithmic growth phase. In one embodiment, the viable cell concentration of Lactobacillus gasseri in the culture product is 25 billion to 200 billion cfu / g, in a preferred embodiment, the viable cell concentration of Lactobacillus gasseri is 37.5 billion to 150 billion cfu / g, in a more preferred embodiment, the viable cell concentration of Lactobacillus gasseri is 50 billion to 100 billion cfu / g, and in the most preferred embodiment, the viable cell concentration of Lactobacillus gasseri is 100 billion cfu / g.

[0012] Since the present invention also relates to the discovery that a novel Lactobacillus gasseri has an effect of improving sleep quality, the present invention relates to a method for improving sleep quality with Lactobacillus gasseri.

[0013] In one embodiment, Lactobacillus gasseri or its culture product of the present invention is prepared as a composition. In a preferred embodiment, the composition is a pharmaceutical composition. In a preferred embodiment, the composition is a food composition. The compositions of the present invention can be obtained by compounding Lactobacillus gasseri or its culture product of the present invention with other well-known pharmaceutically and / or food-acceptable additives by any known technique. These additives include, but are not limited to, carriers, excipients, adhesives, fillers, disintegrants, diluents, lubricants and / or sweeteners or flavorings.

[0014] In one embodiment, the content range of Lactobacillus gasseri or its culture product in the composition can be adjusted according to actual requirements, for example, according to parameters such as the body weight and age of the individual. In a preferred embodiment, the composition contains about 2 billion cfu to about 100 billion cfu of Lactobacillus gasseri or its culture product; in a more preferred embodiment, the composition contains about 5 billion cfu to about 50 billion cfu of Lactobacillus gasseri or its culture product; and in a more preferred embodiment, the composition contains about 20 billion cfu of Lactobacillus gasseri or its culture product.

[0015] In one embodiment, the composition includes other components in addition to Lactobacillus gasseri or its culture product. In one embodiment, the composition includes one or more of the following in addition to Lactobacillus gasseri or its culture product: imidazole dipeptide composition, raffinose oligosaccharide, chitosan oligosaccharide, and inulin. In a preferred embodiment, the composition contains about 10% to about 20% of the imidazole dipeptide composition, and in a more preferred embodiment, the composition contains about 17% of the imidazole dipeptide composition. In a preferred embodiment, the composition contains about 15% to about 25% of the raffinose oligosaccharide, and in a more preferred embodiment, the composition contains about 22.7% of the raffinose oligosaccharide. In a preferred embodiment, the composition contains about 2% to about 8% of the chitosan oligosaccharide, and in a more preferred embodiment, the composition contains about 5.7% of the chitosan oligosaccharide. In one preferred embodiment, the composition contains about 15% to about 20% inulin, and in one more preferred embodiment, the composition contains about 17% inulin. In one embodiment, one or more components of the composition produce a synergistic effect with Lactobacillus gasseri or its culture product.

[0016] In one embodiment, the imidazole dipeptide composition contains 5% to 60% peptide components by weight, and these peptide components include anserine and carnosine. In a preferred embodiment, the imidazole dipeptide composition contains about 5% to 40% anserine and 0.5% to 20% carnosine by weight, and in a more preferred embodiment, the imidazole dipeptide composition contains 10% to 15% peptides, of which about 10% is anserine and about 1% is carnosine.

[0017] In one embodiment, the improvement of sleep quality by the composition of the present invention includes increasing the production of 5-hydroxytryptophan (5-HTP). In a preferred embodiment, the composition increases the production of 5-hydroxytryptamine (5-HT).

[0018] In one embodiment, the improvement of sleep quality by the composition of the present invention includes inhibiting the activity of monoamine oxidase (MAO).

[0019] In one embodiment, the improvement of sleep quality by the composition of the present invention includes reducing the number of awakenings during sleep.

[0020] In one embodiment, the improvement of sleep quality by the composition of the present invention includes reducing the amount of wakefulness during sleep.

[0021] In one embodiment, the improvement of sleep quality by the composition of the present invention includes an increase in the production of 5-hydroxytryptophan (5-HTP) or 5-hydroxytryptamine (5-HT), inhibition of monoamine oxidase (MAO) activity, a reduction in the number of awakenings during sleep, a reduction in the amount of wakefulness during sleep, or one or more of the above.

[0022] The present invention will be described in detail below. Other features, purposes, and advantages of the present invention can be readily found in the specific embodiments and claims of the invention. [Brief explanation of the drawing]

[0023] [Figure 1A] Shows the experimental results of different Lactobacillus strains stimulating an intestinal enterochromaffin cell model to secrete 5-hydroxytryptophan (5-HTP). [Figure 1B] Shows the inhibition rate of fermentation supernatants of different strains against monoamine oxidase (MAO)-A. [Figure 2A] Shows a typical diagram of the movement trajectories of zebrafish after sample treatment, wherein in the diagram, the black line represents low-speed movement distance, the green line represents medium-speed movement distance, and the red line represents high-speed movement distance. [Figure 2B] Shows the amount of wakefulness activity of zebrafish after sample treatment (compared with the model control group, **p<0.01, ***p<0.001). [Figure 3] Shows the MAO-A enzyme inhibition rate of a composition comprising Lactobacillus gasseri and each component thereof. DETAILED DESCRIPTION OF EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0024] Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings that are commonly understood by those skilled in the art. While the meaning and scope of any term should be clear, in the event of any potential ambiguity, the definition provided herein shall take precedence over any dictionary or external definition.

[0025] As used herein, unless otherwise indicated, the following terms shall be understood to have the following meanings.

[0026] Throughout this specification, the term "comprising" should be understood to imply the inclusion of the stated element(s) or group of elements, but does not exclude any other element(s) or group of elements.

[0027] Unless otherwise explicitly defined otherwise in the context, the singular forms "a" and "the" include plural referents. Unless otherwise required by the context, plural terms also include the singular form.

[0028] Numerical values can represent approximate values using the term "about", and can be understood as another aspect of a specific value. When reference is made to other endpoints and the other endpoints per se, it can be further understood that both endpoints of each range are significant. As used herein, "about" represents ±10%.

[0029] In the present invention, the terms "isolated" or "separated" mean that a substance is removed from its original environment (for example, if it exists naturally, the original environment is the natural environment). The terms "isolated" or "separated" do not necessarily refer to a purified substance.

[0030] The term "appropriate conditions" means conditions such as temperature and culture time that allow the growth and propagation of *Lactobacillus gasseri*. A person skilled in the art can adjust the components of a medium and culture conditions based on existing knowledge. Generally, the culture temperature for *Lactobacillus gasseri* is about 20°C to about 45°C, preferably about 30°C to about 40°C, more preferably about 37°C, and the culture time may be more than 10 hours, preferably more than 12 hours, more preferably more than 16 hours.

[0031] The term "amelioration" refers to an approach that achieves beneficial or desired results including clinical outcomes. For the purposes of the present application, beneficial or desired results include, but are not limited to, suppressing and / or inhibiting the onset and / or progression of a medical condition, or reducing the severity of such a medical condition, for example, reducing the number and / or severity of symptoms associated with the medical condition, improving the quality of life of a patient suffering from the medical condition, reducing the dosage of other drugs required for the treatment of the medical condition, and / or enhancing the effect on the medical condition of another type of drug taken by the patient.

[0032] The terms “administer” or “give” a substance, compound, or drug can be used by one of the various methods known to those skilled in the art. For example, a compound or drug can be administered orally. It can also be administered, for example, once, multiple times, and / or over one or more extended periods.

[0033] As used herein, the term "sleep quality" refers to the quality of sleep, and good sleep quality is defined as having sufficient sleep duration (7-8 hours of sleep per day), being able to fall asleep easily (less than 30 minutes), sleeping soundly (sleeping until dawn without waking up in the middle of the night), not being startled awake, and being able to get up easily and refreshed. Referring to the Pittsburgh Sleep Quality Index (PSQI), the assessment of sleep quality includes daytime dysfunction (impairment of daytime functioning due to sleep problems, e.g., sleepiness, difficulty concentrating), use of hypnotics (explaining the frequency and degree of falling asleep with the use of drugs), sleep disorders (e.g., difficulty breathing, coughing or snoring, feeling cold or hot, having bad dreams), sleep efficiency (difficulty falling asleep), sleep duration, and subjective sleep quality.

[0034] The terms “patient,” “individual,” or “person” are interchangeable and refer to humans or non-human animals. These terms include mammals such as humans, primates, domesticated animals (including cattle and pigs), pet animals (e.g., dogs and cats), and rodents (e.g., mice and rats).

[0035] In this invention, we unexpectedly discovered that Lactobacillus gasseri LyLG1 and its culture product can improve sleep quality. After administration of Lactobacillus gasseri LyLG1 culture product, the production of 5-HTP can be effectively promoted, inhibiting the MAO-A enzyme. Animal studies also found that Lactobacillus gasseri LyLG1 has effects such as reducing the number of awakenings during sleep and decreasing the amount of waking activity during sleep. Thus, this invention provides a novel Lactobacillus gasseri and its new use in sleep improvement, offering a novel method for improving sleep quality that replaces or supports conventional small molecule drugs.

[0036] The present invention has been described in general terms above. The present invention can be more easily understood by referring to the following examples, which provide exemplary ways of carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure the precision of the digits used (e.g., quantity, temperature, etc.), but of course, some experimental errors and variations are acceptable. Modifications and changes made to the examples discussed herein without departing from the spirit or category of the present invention still fall within the scope of the present invention.

[0037] Examples Example 1: Screening of Lactobacillus gasseri Different platform screening methods are used to select strains of Lactobacillus gasseri with specific characteristics and functionalities from a variety of strains.

[0038] Example 1.1: A study in which different Lactobacillus gasseri strains stimulate enterochromaffin cell models to secrete 5-HTP. Enterochromaffin RIN14B cell line cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C for approximately 72 hours until the cells reached an appropriate density. After washing the cells with sterile phosphate buffer (PBS), the medium was replaced with serum-free medium (Tian P, Zhu H, Zou R, Kong Q, Xu M, Zhao J, Zhang H, Chen W, Wang G., An in vitro screening method for probiotics with antidepressant-like effect using the enterochromaffin cell model., Food Funct., 2021 Jan 21;12(2):646 - 655. Epub 2021 Jan 6).

[0039] Furthermore, Lactobacillus strains Ly11 (Litong Co., Ltd. Lacticaseibacillus paracasei strain R094), LT12 (Litong Co., Ltd. Lacticaseibacillus paracasei strain LT12, Food Industry Development Institute deposit number: BCRC 910461), Ly17 (Limosilactobacillus fermentum strain CIP102980), LyFD (Litong Co., Ltd. Lactobacillus reuteri), LyLG1 (Litong Co., Ltd. Lactobacillus gasseri strain LyLG1, China Typical Culture Depository Center CCTCC deposit number: M20241476), LyLF (Litong Co., Ltd. Lactobacillus fermentum), and LyT1 (Litong Co., Ltd. Lactobacillus fermentum) were cultured in MRS medium until the logarithmic growth phase, and the bacteria were collected from each strain and the concentration was adjusted to 10. 8 After adjusting the concentration to CFU / mL, the supernatant was collected after filtration through a 0.22 μm filtration membrane.

[0040] The collected bacterial supernatants were co-cultured with RIN14B cells for 1 hour, and after collecting the cell supernatants, the 5-HTP content in the cell supernatants was measured using HPLC fluorescence detection.

[0041] Example 1.2: Study of MAO crude enzyme inhibitory activity by different Lactobacillus gasseri strains Crude monoamine oxidase (MAO) enzyme was extracted from the liver of aged male rats. Liver tissue was washed with pre-cooled PBS buffer, connective tissue was removed, and the tissue was chopped. Homogenization was performed by adding pre-cooled 0.25 mol / L sucrose solution. Sequential centrifugation was performed at low speed (1000 g, 4°C) and high speed (10000 g, 4°C), discarding the supernatant and precipitate and retaining the intermediate layer. The intermediate layer was suspended in 1.2 mol / L sucrose solution and subjected to ultra-high-speed centrifugation for 2 hours (53000 g, 4°C), and the precipitate was collected. The precipitate was washed with 1.15% KCl and 0.02 mol / L PBS, and finally the crude enzyme was suspended in PBS, aliquoted, and stored at -80°C.

[0042] Lactobacillus fermentation broth was prepared, and the seven different Lactobacillus strains listed above were activated in MRS medium for 24 hours to obtain single colonies. These were inoculated at a 4% dose into 10% skim milk medium and cultured at 37°C for 48 hours to obtain fermentation broth. The fermentation broth was collected and subjected to a 10-minute boiling water bath to inactivate the cells and enzymes. Centrifugation and filtration were performed to remove large molecular impurities such as cells and coagulation proteins to obtain a cell-free fermentation supernatant, which was freeze-dried for use.

[0043] MAO activity inhibition rates were measured. 110 μL of PBS buffer, 20 μL of crude MAO enzyme solution, and 40 μL of the test sample (fermentation supernatant) were added to a 96-well plate, mixed homogeneously, and incubated at 37°C for 5 minutes. 30 μL of kynuramine substrate (1 mmol / L) was added, mixed homogeneously by shaking, and absorbance A1 (before reaction) was measured at a wavelength of 360 nm using an ELISA. After reacting at 37°C for 60 minutes, absorbance A2 (after reaction) was measured again at a wavelength of 360 nm using the ELISA. PBS buffer was used as a blank control, and absorbance A0 was measured. The formula for calculating the relative inhibition rate (%) of MAO is [1-(A1-A2) / A0]×100% (Huang Qi-wang, You Chun-ping, Chen Ping-jing. Measurement of monoamine oxidase activity inhibition rate by lactic acid bacteria fermentation liquid. Applied Chemical Industry, 2016, 45(12):2390-2394. 2016.12.005).

[0044] The fermentation supernatants of the seven different Lactobacillus strains mentioned above were measured, and the results are shown in Figure 1B.

[0045] Using the two measurement platforms described above, Lactobacillus gasseri LyLG1 was co-cultured with a cell model for one hour, and the 5-HTP content of the supernatant was measured. The results showed that the 5-HTP content was significantly higher than that of other test strains, indicating that Lactobacillus gasseri LyLG1 can effectively stimulate enterochromaffin cell models to secrete 5-HTP. Furthermore, after fermenting Lactobacillus gasseri LyLG1 for 24 hours, the fermented supernatant was measured, and it was found that the fermented supernatant could also effectively inhibit the MAO-A enzyme, and that the inhibitory effect was superior to that of other test strains.

[0046] Example 2: Study on the sleep-improving effects of Lactobacillus gasseri LyLG1 and its composition. Of the seven bacterial strains tested, Lactobacillus gasseri LyLG1 showed the most superior efficacy, and therefore, Lactobacillus gasseri LyLG1 was selected as the strain to be used in subsequent experiments.

[0047] Furthermore, Lactobacillus gasseri LyLG1 was prepared into a composition (hereinafter referred to as "Composition X"), and the specific ratios of the ingredients are shown in Table 1 below.

[0048] [Table 1]

[0049] Example 2.1: Measurement of the maximum measurable concentration (MTC) of Lactobacillus gasseri LyLG1 and composition X Wild-type AB zebrafish 5 days post-fertilization (5 dpf) were randomly selected and placed in a 6-well plate. Each well (experimental group) contained 30 zebrafish. Lactobacillus gasseri LyLG1 samples dissolved in water (see Table 2 for concentrations) were administered. Simultaneously, a normal control group and a model control group were established, with each well containing 3 mL. After treatment at 28°C for 1 day, each experimental group except the normal control group was administered pentetrazol dissolved in water to establish a zebrafish insomnia model. After continuing treatment for 1 hour, the MTC of the zebrafish model using the samples was measured. Under these experimental conditions, the MTC of the sleep-improving effect of composition X and Lactobacillus gasseri LyLG1 was 1000 μg / mL for both. See Table 2 for details.

[0050] [Table 2]

[0051] Example 2.2: Evaluation of the sleep-improving effects of Lactobacillus gasseri LyLG1 and composition X 5dpf wild-type AB zebrafish were randomly selected and placed in a 6-well plate, with each well (experimental group) containing 30 zebrafish. A sample dissolved in water (concentration shown in Table 3) was administered. The positive control group received 29.0 μg / mL of oxazepam. Simultaneously, a normal control group and a model control group were established, with each well containing 3 mL. After 1 day of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and transferred to a 96-well plate, with 200 μL of sample and one fish placed in each well. All experimental groups except the normal control group were administered pentetrazole dissolved in water to establish a zebrafish insomnia model. The amount of arousal activity in the zebrafish within 1 hour was measured using a behavioral analyzer (ZebraLab3.22.3.31, Viewpoint, France), and the sleep-improving effect of the samples was evaluated based on the statistical analysis results of the above indicators. Statistical analysis results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and a p<0.05 value indicates that the difference is statistically significant.

[0052] Under these experimental conditions, the effective concentration of oxazepam was 29.0 μg / mL, corresponding to the daily human intake of 174 mg. The sleep-improving effect of composition X and LyLG1 Lactobacillus gasseri is specifically expressed as a reduction in the amount of arousal activity in zebrafish, with composition X and LyLG1 Lactobacillus gasseri showing effects at 500 μg / mL and 1000 μg / mL, respectively. For details of the experimental results, please refer to Table 3, Figure 2A, and Figure 2B.

[0053] [Table 3]

[0054] In this experiment, we used a fungal powder produced by fermentation in the laboratory, resulting in a bacterial count of 3.0 × 10⁶. 10 The cfu / g value was measured, the amount of Lactobacillus gasseri LyLG1 added to composition X was 37.5%, and the bacterial count in composition X was 1.125 × 10⁶. 10It can be seen that the concentration is cfu / g.

[0055] At a concentration of 500 μg / mL, the amount of Lactobacillus gasseri LyLG1 added to composition X is approximately 5.625 × 10⁻⁶. 6 The concentration was cfu / mL, and the bacterial count in the Lactobacillus gasseri LyLG1 experimental group was approximately 1.69 × 10⁶. 7 This is cfu / mL, and when converted to a daily dose for the human body, Composition X: Approximately 3.4 × 10 10 cfu Lactobacillus gasseri LyLG1: approx. 9.0 x 10 10 cfu.

[0056] When the number of Lactobacillus gasseri LyLG1 bacteria added to composition X is small, it can be concluded that the effect of pure bacterial powder can be achieved, or even surpassed, and it can be considered that the other components in the formulation assist Lactobacillus gasseri LyLG1, resulting in a synergistic effect.

[0057] Example 3: In vitro experiment of MAO-A enzyme inhibition rate To determine whether each component in composition X has additional effects, MAO-A enzyme inhibition rate experiments were conducted on several components of composition X that are presumed to have effects on sleep aid and relief of nervous tension, at the same concentration as composition X itself. The zebrafish experiment verified that composition X has a synergistic effect that is more pronounced when the bacterial count is low.

[0058] In the experiment, recombinant human MAO-A (purchased from Sigma Aldrich, product number M7316) and quinulamine were used as the substrate. The enzyme reaction mixture, containing 100 mM phosphate buffer (pH 7.4), 250 μM quinulamine, MAO-A enzyme (0.1 mg / ml), and the sample, was reacted at 37°C in the dark for 40 minutes. After the reaction was complete, Amplex Red (0.5 mM) and HRP (1 mg / mL) were added to the mixture and the reaction was continued for 5 minutes. Sulfuric acid (0.5 M) was added to the mixture to terminate the reaction, and the absorbance at 571 nm was measured using a spectrophotometer.

[0059] The inhibition rate of MAO-A enzyme for each component of composition X was measured, and the relevant data for each component group are as follows. In this experiment, the concentration for each group was 17.6 mg / ml.

[0060] Group 1 Composition X: See Table 1 Group 2 Inulin: 100% Inulin Group 3: Lactobacillus gasseri LyLG1 and Inulin: 37.5% Lactobacillus gasseri and 62.5% Inulin Group 4 Imidazole dipeptide composition and inulin: 17% imidazole dipeptide composition and 83% inulin Group 5 Lactobacillus gasseri LyLG1: 100% Lactobacillus gasseri Group 6 Imidazole dipeptide composition: 100% imidazole dipeptide composition (containing peptides in a quantity of 10% to 15%, of which anserine is approximately 10% and carnosine is approximately 1%).

[0061] The experimental results are shown in Figure 3. From Figure 3, it can be seen that the MAO enzyme inhibition rate of composition X at this concentration is far higher than that of each individual component group. In this experiment, the imidazole dipeptide composition components themselves showed a certain degree of MAO enzyme inhibition, but the inhibition rate of the 100% concentration imidazole dipeptide composition (group 6) against the MAO enzyme was far lower than that of composition X (group 1), which contained only 17% of the imidazole dipeptide composition.

[0062] Example 4: Genetic information analysis of LyLG1 Lactobacillus gasseri LyLG1 Lactobacillus gasseri is obtained as follows: The skins are removed from soybeans and they are boiled thoroughly. The strain is inoculated, wrapped in banana leaves, and fermented for 36-48 hours to obtain a white or yellowish paste-like substance, after which the fermentation liquid is extracted. After diluting the fermentation liquid, it is spread onto standard MRS agar plates and incubated for 24 hours. A plate with an appropriate colony growth density is selected, a single colony is selected, and the strain is isolated and purified by streaking culture. The purified strain is then placed in MRS agar containing 20% ​​glycerol to prepare a glycerol cryovial, which is stored frozen at -80°C.

[0063] rRNA gene sequencing was performed on the isolate using a method combining the third-generation PacBio and second-generation Illumina technologies (Guangzhou Jidiao Biotechnology Co., Ltd.), and the strain was identified as Lactobacillus gasseri. Some of the 16S rRNA sequences are as follows:

[0064] [Table 4] TIFF2026142975000006.tif75149

[0065] [Biological material deposit] Domestic deposit information [Note the depositary institution, date, and number in that order] Food Industry Development Research Institute, September 18, 2024, BCRC 911242 Information on overseas deposits [Note the depositing country, institution, date, and number in that order] China, China Center for Type Culture Collection, CCTCC, July 3, 2024, M20241476

Claims

1. An isolated strain of Lactobacillus gasseri, wherein the Lactobacillus gasseri strain has substantially the same characteristics as the strain with deposit number BCRC 911242 deposited with the Food Industry Development Research Institute, and the strain contains the sequence SEQ ID NO:

1.

2. The strain according to claim 1, which is LyLG1 with deposit number BCRC 911242 deposited with the Food Industry Development Research Institute.

3. A method for producing a culture product of Lactobacillus gasseri, comprising inoculating a strain described in claim 1 or 2 into a suitable culture medium and culturing the culture under appropriate conditions to obtain the culture product.

4. The method according to claim 3, wherein the culture medium is a solid culture medium or a liquid culture medium.

5. The method according to claim 4, wherein the culture medium is MRS medium.

6. The method according to any one of claims 3 to 5, wherein the viable cell concentration of Lactobacillus gasseri in the culture product is 25 billion to 200 billion cfu / g.

7. The method according to any one of claims 3 to 5, wherein the viable cell concentration of Lactobacillus gasseri in the culture product is 37.5 billion to 150 billion cfu / g.

8. The method according to any one of claims 3 to 5, wherein the viable cell concentration of Lactobacillus gasseri in the culture product is 50 billion to 100 billion cfu / g.

9. The method according to any one of claims 3 to 5, wherein the viable cell concentration of Lactobacillus gasseri in the culture product is 100 billion cfu / g.

10. A culture product of Lactobacillus gasseri produced by the method according to any one of claims 3 to 9.

11. A composition comprising the strain described in claim 1 or 2 or the culture product described in claim 10.

12. The composition according to claim 11, comprising one or more of an imidazole dipeptide composition, a raffinose oligosaccharide, a chitosan oligosaccharide, and inulin.

13. The composition according to claim 12, wherein the imidazole dipeptide composition comprises 5% to 60% by weight of peptide components, and the peptide components comprise anserine and carnosine.

14. The composition according to claim 13, wherein the imidazole dipeptide composition comprises about 5% to 40% anserine and 0.5% to 20% carnosine.

15. The composition according to claim 14, wherein the imidazole dipeptide composition comprises peptides in an amount of 10% to 15%, of which anserine accounts for about 10% and carnosine accounts for about 1%.

16. The composition according to any one of claims 11 to 15, comprising about 10% to about 20% of an imidazole dipeptide composition, about 15% to about 25% of raffinose oligosaccharide, about 2% to about 8% of chitosan oligosaccharide, and about 15% to about 20% of inulin.

17. The composition according to any one of claims 11 to 15, comprising about 17% imidazole dipeptide composition, about 22.7% raffinose oligosaccharide, about 5.7% chitosan oligosaccharide, and about 17% inulin.

18. Use of the strain according to claim 1 or 2, the culture product according to claim 10, or the composition according to any one of claims 11 to 17 for producing a composition for improving sleep quality.

19. The use according to claim 18, wherein the composition is a pharmaceutical composition or a food composition.

20. The use according to claim 18 or 19, wherein the improvement in sleep quality includes an increase in the production of 5-hydroxytryptophan (5-HTP) or 5-hydroxytryptamine (5-HT), inhibition of monoamine oxidase (MAO) activity, a reduction in the number of awakenings during sleep, a reduction in the amount of wakefulness during sleep, or a combination of one or more of the above.

21. The use according to claim 18 or 19, wherein the composition comprises about 2 billion cfu to about 100 billion cfu of the strain or its culture product.

22. The use according to claim 18 or 19, wherein the composition comprises about 5 billion cfu to about 50 billion cfu of the strain or its culture product.

23. The use according to claim 18 or 19, wherein the composition comprises approximately 20 billion cfu of the bacterial strain or its culture product.

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