Lactobacillus helveticus for modulating biological circadian rhythms by producing s-adenosylmethionine

Abstract

The application discloses a lactobacillus helveticus for regulating biological circadian rhythm by producing S-adenosylmethionine, and belongs to the field of microorganisms. The lactobacillus helveticus CCFM1320 provided by the application can improve hyperactivity behavior caused by sleep deprivation, relieve cognitive and memory function abnormalities, promote normal expression of circadian rhythm related genes, and improve serum metabolite and brain neurotransmitter disorder. It is helpful to provide more personalized treatment programs for sleep disorders in the future, and has very promising application prospect.

Description

Technical Field

[0001] This invention relates to a type of Lactobacillus helveticus that regulates biological diurnal rhythms by producing S-adenosylmethionine, belonging to the field of microbial technology. Background Technology

[0002] Sleep deprivation has become a widespread phenomenon in modern society, with most people unable to meet their daily sleep needs. Sleep deprivation leads to circadian rhythm disruption and abnormal sleep / wake cycles, negatively impacting important functions such as neurodevelopment, learning, memory, and mood regulation. The mechanism is related to neurophysiological changes in the prefrontal cortex. Circadian rhythm disturbances have numerous negative effects on brain function. In mammals, long-term circadian rhythm disruption can lead to many diseases, such as cardiometabolic syndrome, obesity, and psychosomatic health disorders.

[0003] Circadian rhythms are regulated by various neurotransmitters, among which melatonin is particularly important. Melatonin synthesis relies on S-adenosylmethionine (S-Amethionine). In the pineal gland of the brain, S-A-Amethionine promotes the methylation of N-acetylserotonin into melatonin; abnormal S-A-Amethionine levels can affect the normal synthesis of melatonin in the brain. Currently, the main methods for synthesizing S-A-Amethionine include chemical synthesis, microbial fermentation, in vitro enzymatic synthesis, and whole-cell catalysis, with microbial fermentation being the primary pathway. Studies have shown that probiotics can also produce S-A-Amethionine, and several probiotic strains producing S-A-Amethionine have been reported, including one strain of *Bifidobacterium bifidum*. BGN4 The highest yield was achieved at 0.22 nmol / mL.

[0004] Recent studies have found a strong correlation between gut microbiota dysbiosis and circadian rhythm disruption. With the proposal of the "microbe-gut-brain axis" hypothesis, it has become possible to regulate brain function by modulating the gut microbiota. The microbiota can influence the brain in multiple ways; its metabolites affect the host's learning, memory, and cognitive functions. As an important dietary supplement for regulating the gut microbiota, it shows great potential in improving nervous system function. However, there is currently no direct evidence that probiotics improve circadian rhythm disruption caused by sleep deprivation (or sleep disorders) by producing S-adenosylmethionine. Summary of the Invention

[0005] This invention provides a strain of Lactobacillus helveticus ( Lactobacillus helveticus The Lactobacillus helveticus CCFM1320 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCCNO:63716.

[0006] The Lactobacillus helveticus CCFM1320 is a Gram-positive bacterium that appears as a rod under a microscope. When grown on MRS plates, it forms smooth, translucent, round colonies that are white with neat edges. In MRS liquid medium, it grows in a uniformly turbid manner, and the cells precipitate white after prolonged storage. The optimal growth temperature is 37°C.

[0007] The present invention also provides a microbial preparation containing the aforementioned Lactobacillus helveticus CCFM1320.

[0008] In one embodiment, the amount of *Lactobacillus helveticus* CCFM1320 added to the microbial preparation is not less than 5 × 10⁻⁶. 9 CFU / g or 5×10 9 CFU / mL.

[0009] In one embodiment, the microbial preparation is a solid or liquid preparation.

[0010] In one embodiment, the microbial preparation is a powder obtained by drying a bacterial solution containing Lactobacillus helveticus CCFM1320.

[0011] In one embodiment, the drying refers to vacuum freeze drying.

[0012] The present invention also provides the use of the Lactobacillus helveticus CCFM1320 or the microbial preparation in the preparation of a drug containing the function of regulating circadian rhythm.

[0013] In one embodiment, the drug can treat circadian rhythm disorders caused by sleep deprivation.

[0014] In one embodiment, the amount of *Lactobacillus helveticus* CCFM1320 added to the drug is not less than 5 × 10⁻⁶. 9 CFU / g or 5×10 9 CFU / mL.

[0015] In one embodiment, the drug contains the Lactobacillus helveticus CCFM1320, a drug carrier, and / or pharmaceutical excipients.

[0016] In one embodiment, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0017] In one embodiment, the dosage form of the drug is granules, capsules, tablets, pills, or oral liquid.

[0018] In one implementation, the application includes at least one of the following functions: (a) Regulating the circadian rhythm of organisms; (b) Improve hyperactivity caused by sleep disorders; (c) Improve exploration and memory impairment caused by sleep disorders.

[0019] The present invention also provides a drug for regulating circadian rhythms, the drug containing the aforementioned Lactobacillus helveticus CCFM1320.

[0020] In one embodiment, the amount of *Lactobacillus helveticus* CCFM1320 added to the drug is not less than 5 × 10⁻⁶. 5 CFU / g or 5×10 9 CFU / mL.

[0021] This invention also provides the application of the above-mentioned Lactobacillus helveticus CCFM1320 in the preparation of a drug or functional food having at least one of the following functions: (a) Alleviates the decrease in serum S-adenosylmethionine levels caused by sleep deprivation; (b) Alleviate the reduction in melatonin receptor synthesis and melatonin levels in the brain caused by sleep deprivation; (c) Alleviate hyperactivity and impaired exploration and memory functions caused by sleep deprivation; (d) Alleviate the abnormal expression of hypothalamic circadian rhythm-related genes caused by sleep deprivation.

[0022] The present invention also provides the application of the aforementioned Lactobacillus helveticus CCFM1320 in the preparation of food.

[0023] In one embodiment, the food is a dairy product, soy product, or fruit and vegetable product produced using Lactobacillus helveticus CCFM1320 or a fermentation agent of the above-mentioned microbial preparation.

[0024] In one embodiment, the dairy products include fermented milk, flavored fermented milk, fermented milk beverages, cream, cheese, milk-containing beverages, or milk powder; the soy products include soy milk and soy milk powder; and the fruit and vegetable products include fruit and vegetable products made from at least one of cabbage, white radish, cucumber, beet, yellow peach, or bayberry products.

[0025] In one embodiment, the food is a fermented food, including solid food, liquid food, or semi-solid food.

[0026] In one embodiment, the food contains the Lactobacillus helveticus CCFM1320.

[0027] Beneficial Effects: This invention provides a novel application of *Lactobacillus helveticus* CCFM1320 in the prevention and / or alleviation of circadian rhythm disorders caused by sleep deprivation. *Lactobacillus helveticus* CCFM1320 has a high capacity for producing S-adenosylmethionine (S-methionine). Intervention with sleep-deprived mice using *Lactobacillus helveticus* CCFM1320 increased serum S-methionine levels, and by regulating melatonin synthesis in the brain, restored the normal expression of circadian rhythm-related genes in the brains of sleep-deprived mice, reducing hyperactivity, exploratory behavior, and memory / cognitive deficits. Controlled experiments demonstrated that *Lactobacillus helveticus* with low S-methionine production (DSCAB10M13) did not have this effect. This invention demonstrates that *Lactobacillus helveticus* with high S-methionine production has the effect of regulating circadian rhythms and improving sleep. This invention expands the application scope of *Lactobacillus helveticus* as a probiotic and provides theoretical guidance for the prevention, alleviation, and treatment of neurological dysfunction using probiotics.

[0028] Preservation of biological materials A strain of Lactobacillus helveticus ( Lactobacillus helveticus CCFM1320, its taxonomic name is: Lactobacillus helveticus It was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 63716, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0029] Figure 1 The yield of S-adenosylmethionine by CCFM1320; where ****P<0.0001.

[0030] Figure 2 The effect of CCFM1320 on the abnormal serum S-adenosylmethionine content in sleep-deprived mice was investigated; *P<0.05, ****P<0.0001.

[0031] Figure 3 To investigate the regulatory effect of CCFM1320 on melatonin levels and abnormal melatonin receptor synthesis in the brains of sleep-deprived mice; (A) melatonin levels in the striatum; (B) expression level of MT1 mRNA in the hypothalamus; (C) expression level of MT2 mRNA in the hypothalamus; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Figure 4 The results are the performance of mice in behavioral experiments; (A) open field test; (B) new object recognition test; (C) Y-maze test; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0033] Figure 5 The regulatory effect of CCFM1320 on the expression of genes related to circadian rhythms in sleep-deprived mice; (A) hypothalamus Per1 (A) mRNA expression levels; (B) in the hypothalamus Per2 mRNA expression levels; (C) in the hypothalamus Bmal1 mRNA expression levels; where *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0034] The Lactobacillus helveticus CCFM1320 of this invention was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC NO.63716.

[0035] Determination of S-adenosylmethionine content: Mobile phase: A: 0.01 mol / L ammonium formate, B: methanol; Elution conditions: isocratic elution, flow rate 1 mL / min; Column temperature: 30℃; Wavelength: 254 nm; Column: X Bridge C18 (5 μm, 4.6 mm × 250 mm).

[0036] Example 1: Screening of Lactobacillus helveticus and preparation of bacterial suspension (a) Isolation and screening of lactobacilli: (l) Take 1g of fresh feces from a healthy adult. Dilute it serially and spread it on MRS solid medium, then incubate at 37°C for 72 hours; (2) Observe and record the colony morphology, and pick colonies for streaking and purification; (3) In MRS liquid medium, culture at 37°C for 48 hours, and then Gram stain the resulting colonies to record the colony morphology.

[0037] (4) Discard the Gram-negative bacterial strains and Gram-positive cocci in the colonies and select the Gram-positive bacilli.

[0038] (5) After catalase analysis, discard catalase-positive strains and retain catalase-negative strains.

[0039] (II) Preliminary identification of lactobacilli (1) The gelatin liquefaction test result was negative; (2) The indole test result was negative; (3) The hydrogen sulfide test result was negative; (4) The nitrate reduction test result was negative; (5) The catalase test result was negative; (6) The glucose gas production test result was negative.

[0040] Based on the above results, the preliminary diagnosis is lactobacillus.

[0041] (III) Molecular biological identification of Lactobacillus: (l) Single-strain genome extraction: The Lactobacillus strains screened in step (ii) were cultured overnight. 1 mL of the overnight bacterial suspension was transferred to a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 2 min. The supernatant was discarded to obtain bacterial cells. The bacterial cells were washed with 1 mL of sterile water and centrifuged at 10,000 rpm for 2 min. The supernatant was discarded to obtain bacterial cells. 200 μL of the supernatant was added to the centrifuge tube. Incubate SDS lysis buffer at 80°C for 30 min. Add 200 μL of phenol-chloroform solution to the bacterial lysis buffer, where the composition and volume ratio of the phenol-chloroform solution is Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1. Mix thoroughly by inversion, centrifuge at 12000 rpm for 5-10 min, and collect 200 μL of the supernatant. Add 400 μL of ice-cold ethanol or ice-cold isopropanol to 200 μL of the supernatant, incubate at -20°C for 1 h, centrifuge at 12000 rpm for 5-10 min, and discard the supernatant. Resuspend the precipitate in 500 μL of 70% (v / v) ice-cold ethanol, centrifuge at 12000 rpm for 1-3 min, and discard the supernatant. Dry in a 60°C oven or air dry. Redissolve the precipitate in 50 μL of ddH2O for PCR. (2) 16S rDNA PCR: A. Bacterial 16S rDNA 50μL PCR reaction system: 10×Taq buffer, 5μL; dNTP, 5μL; 27F, 0.5μL; 1492R, 0.5μL; Taq enzyme, 0.5μL; template, 0.5μL; ddH2O, 38μL.

[0042] B. PCR conditions: 95℃ 5min; 95℃ 10s; 55℃ 30s; 72℃ 30s; step2-4 30×; 72℃ 5min; 12℃ 2min; C. Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging; D. The obtained PCR product was sent to a professional sequencing company. The sequencing results were compared with those obtained by searching and similarity analysis in GeneBank using BLAST, and it was identified as Lactobacillus helveticus.

[0043] (3) Whole genome sequencing The extracted whole genome was sent to a professional sequencing company, where a next-generation sequencer was used to sequence the bacterial genome. The obtained sequence results were then searched and compared for similarity in GenBank using BLAST. The sequencing results identified it as a newly discovered strain belonging to *Lactobacillus helveticus*. The strain was stored at -80℃ for later use.

[0044] Preparation method of Lactobacillus helveticus CCFM1320 bacterial agent: Activated second-generation Lactobacillus helveticus CCFM1320 was cultured in MRS liquid medium at 37℃ for 24 h. The bacterial cells were collected by centrifugation at 6000×g for 3 min at 4℃. The supernatant was discarded, and the bacterial cells were resuspended in 10% sterile defatted emulsion to achieve a bacterial concentration of 5×10⁻⁶. 9 CFU / mL.

[0045] Example 2: Lactobacillus helveticus CCFM1320 has the ability to produce high levels of S-adenosylmethionine. Lactobacillus helveticus CCFM1320 was cultured in MRS medium at 37°C for 24 h. The cells were collected and placed in a culture dish containing 0.5 mol / L formic acid, allowed to stand for 1 hour, and the formic acid extract was collected. Cells and debris were removed by centrifugation, and the supernatant was lyophilized. The lyophilized extract was dissolved in 200 µL of HPLC-grade water, filtered through a 0.22 µm filter, and the content of S-adenosylmethionine in the extract was determined by high-performance liquid chromatography (HPLC). Qualitative analysis was based on peak retention time, and quantitative analysis was based on peak area using the external standard method.

[0046] The results show that ( Figure 1 The amount of S-adenosylmethionine produced by CCFM1320 (375.4 μg / L) is approximately the same as that of low-S-adenosylmethionine-producing Lactobacillus helveticus screened using the same screening method. Lactobacillus helveticus The AU of DSCAB10M13 (153.2 μg / L) was more than twice that of the standard. Using the spiking method, the standard had the highest AU, and the sample had the lowest AU. After mixing the standard and the sample 1:1, it was found that the AU of the mixed sample was between the highest and lowest, while the AU of the other peaks in the mixed sample was lower than that of the sample, proving that the sample contained S-adenosylmethionine.

[0047] Example 3: Lactobacillus helveticus CCFM1320 increases serum S-adenosylmethionine levels in sleep-deprived mice The preparation method of Lactobacillus helveticus inoculum is the same as in Example 1.

[0048] Thirty-two male SPF-grade C57BL / 6J mice, 8 weeks old and weighing 22±2g, were selected and housed in an environment with a constant room temperature of 22±2℃ and humidity of 50±5%, with free access to food and water under a 12-hour light / dark cycle. After acclimatization for one week, the mice were divided into groups for treatment, which lasted for 15 days. The grouping and treatment methods are shown in Table 1.

[0049] Table 1. Grouping and treatment methods in animal experiments

[0050] Sleep deprivation animal model: The sleep deprivation animal model is a widely used animal model for investigating circadian rhythms and sleep disorders. A modified multi-platform aquatic environment method was used to construct a mouse sleep deprivation model. The sleep deprivation group, CCFM1320 intervention group, and control bacterial intervention group were treated according to this method (gavage volume: 0.2 mL / mouse / day). The procedure was as follows: Mice were placed in a water tank with a small platform from 13:00 to 9:00 (the next day) to ensure they could not fall asleep, for 15 consecutive days. The blank control group was placed in a water tank with a large platform from 13:00 to 9:00 (the next day), the area of ​​which ensured the mice could fall asleep normally. During this period, the mice could eat and drink independently. On the 15th day, the mice were sacrificed, their venous blood was collected, and serum was obtained by centrifugation. The S-adenosylmethionine content in the serum was measured using an ELISA kit.

[0051] S-adenosylmethionine (S-ADM) plays a crucial role in various biological responses, such as transmethylation, polyamine synthesis, and transsulfation. During methylation, the methyl group of S-ADM is donated to various receptor substrates, such as DNA, phospholipids, and proteins; this process is closely related to circadian rhythms. Studies have shown that inhibiting methylation in mouse and human cells disrupts their circadian rhythms.

[0052] The results showed that ( Figure 2 Compared to the normal control group (1237 pg / mL), sleep deprivation (1155 pg / mL) reduced serum S-adenosylmethionine concentration by 7% (P<0.0001), indicating that sleep deprivation leads to a decrease in serum S-adenosylmethionine levels, resulting in circadian rhythm disruption. CCFM1320 intervention (1197 pg / mL) increased serum S-adenosylmethionine levels in mice by 4% compared to the model group (P<0.05), returning to near-normal levels. However, intervention with Lactobacillus helveticus, which produces low levels of S-adenosylmethionine, resulted in a serum S-adenosylmethionine level of 1149 pg / mL in mice, a decrease of 0.5% compared to the model group (P=0.9716), indicating a worse effect. This indicates that CCFM1320 intervention can increase the serum S-adenosylmethionine content in sleep-deprived mice, thereby maintaining the body's normal methylation level and normal circadian rhythm, while Lactobacillus helveticus, which produces low levels of S-adenosylmethionine, has no effect on the serum S-adenosylmethionine content in mice.

[0053] Example 4: Lactobacillus helveticus CCFM1320 increases melatonin and melatonin receptor levels in the brains of sleep-deprived mice. An animal model was established according to the method in Example 2. Mice were sacrificed on day 15, and mouse brain tissue was collected. The striatum was isolated on ice. The striatum was removed and weighed. The tissue sample was added to 0.4 mol / L perchloric acid solution at a ratio of 1:9 (m:v) and allowed to stand for 10 min to precipitate proteins. Then, the tissue was homogenized using a high-throughput tissue homogenizer and centrifuged at 12000×g, 4℃ for 15 min. 200-300 μL of the supernatant was transferred to a brown sample vial. The melatonin content in the mouse striatum was detected by high-performance liquid chromatography. The retention time of the peak was used for qualitative analysis, and the peak area was measured using the external standard method for quantitative analysis.

[0054] A certain mass of fresh hypothalamic cortex tissue from mouse brain was homogenized with 1 mL of TRIzol and two zirconium beads. Total RNA was extracted using standard methods. The reverse transcription concentration was adjusted to 1 μg / μL, and the purity (A) was determined. 260 / A 280 The range was 1.8-2.0. cDNA was synthesized via reverse transcription and then subjected to real-time quantitative PCR. The sample was mixed with the fluorescent dye SYBR Green Super Mix. The PCR system consisted of 5 μL mix, 1 μL cDNA, and 0.5 μL of forward and reverse primers, with dd H2O added to a total volume of 10 μL. The reaction was performed using a CFX384 real-time quantitative gene amplification instrument. TM The assay was performed on a Real-Time System (Bio-Rad, USA), with three parallel wells for each sample, and housekeeping genes were used. GAPDH This serves as an internal control. The primers used are shown in Table 2. After the reaction, the amplification cycle number (Cq) was calculated and analyzed; the relative expression level was expressed as the fold increase of the target gene relative to the housekeeping gene (2^3). -ΔΔCq ) represents, where -ΔΔCq=(Cq 目的基因 -Cq 管家基因 ) 实验组 -(Cq 目的基因 -Cq 管家基因 ) 对照组 .

[0055] Table 2 Primers

[0056] S-Adenosylmethionine (S-Amethionine) also plays a crucial role in the production of important neurotransmitters in the brain. S-A-Amethionine crosses the blood-brain barrier and enters the brain, where it promotes the conversion of N-acetylserotonin to melatonin in the pineal gland. Melatonin secretion is a prominent endocrine manifestation of the circadian rhythm. Melatonin is an indole hormone secreted by the pineal gland. Recent experimental studies have shown that melatonin regulates the biological clock and stabilizes the body's circadian rhythm, significantly impacting sleep disorders caused by circadian rhythm disturbances. Specifically, this manifests as a shortened sleep latency; a reduced number of awakenings during sleep; a shortened light sleep period and a prolonged deep sleep stage; and a decreased frequency of early awakenings.

[0057] Melatonin plays a crucial role in regulating the body's circadian rhythm and maintaining stable sleep patterns by acting on melatonin receptors (MTs). The MT family consists of two highly conserved structural sequence members: MT1 and MT2 receptors. In the brain, MT1 is mainly expressed in the REM region in important neural functional areas such as the locus coeruleus and the lateral hypothalamus, and is involved in regulating the rapid eye movement (REM) sleep stage of alertness. MT2 is mainly synthesized and secreted in the reticular thalamus (NREM region) and can selectively regulate the non-rapid eye movement (NREM) sleep stage.

[0058] The results showed that ( Figure 3 Compared to the normal control group (0.5901 μg / L), sleep deprivation (0.5652 μg / L) reduced striatal melatonin levels by 5% (P<0.0001), indicating that sleep deprivation leads to a decrease in striatal melatonin levels in mice. Since melatonin secretion has a circadian rhythm, the abnormal striatal melatonin levels in the sleep-deprived group indicate a disruption of this circadian rhythm, likely due to abnormal serum S-adenosylmethionine levels. CCFM1320 intervention (0.5805 μg / L) increased striatal melatonin levels in mice by 3% compared to the model group (P<0.01), returning to near-normal levels. In contrast, after intervention with the control bacteria, the striatal melatonin level in mice was 0.5662 μg / L, only 0.1% higher than the model group (P=0.9914), showing no significant difference. The striatal melatonin level in the CCFM1320 group was 2.5% higher than that in the control bacteria group.

[0059] Compared to the normal control group (MT1 mRNA relative expression level 1.345; MT2 mRNA relative expression level 1.073), sleep deprivation (MT1 mRNA relative expression level 0.782; MT2 mRNA relative expression level 0.528) led to a 42% decrease in the relative expression level of the hypothalamus MT1 gene (P<0.01) and a 51% decrease in the relative expression level of the hypothalamus MT2 gene (P<0.01). This indicates that sleep deprivation leads to abnormal melatonin receptor synthesis and impaired circadian rhythms in mice. CCFM1320 intervention (MT1 mRNA relative expression level 1.205; MT2 mRNA relative expression level 0.960) increased the relative expression level of MT1 gene in the mouse hypothalamus by 54% compared with the model group (P<0.05); and increased the relative expression level of MT2 gene in the mouse hypothalamus by 82% compared with the model group (P<0.05), returning to a near-normal level. Control bacterial intervention (MT1 mRNA relative expression level 0.818; MT2 mRNA relative expression level 0.732) increased the relative expression level of MT1 gene in the mouse hypothalamus by 4.5% compared with the model group (P=0.83); and increased the relative expression level of MT2 gene in the mouse hypothalamus by 35% compared with the model group (P=0.21), with no significant difference. The MT1 mRNA level in the CCFM1320 group was 47% higher than that in the control bacterial intervention group, and the MT2 mRNA level was 31% higher than that in the control bacterial intervention group.

[0060] The results showed that CCFM1320 intervention could increase the melatonin content in the striatum of sleep-deprived mice and increase the relative expression levels of MT1 mRNA and MT2 mRNA in the hypothalamus of mice, promoting the normal synthesis of melatonin receptors in the hypothalamus and maintaining the normal circadian rhythm of the organism. The control group did not have this effect.

[0061] Example 5: Lactobacillus helveticus CCCFM1320 improves hyperactivity, exploration, and memory behaviors in sleep-deprived mice. An animal model was established using the method described in Example 3, and behavioral experiments were conducted on it.

[0062] Open field: The experiment was conducted in a quiet environment. The animal was placed in the center of the bottom of the box, and video recording and timing were performed simultaneously. Video recording was stopped after 5 minutes of observation. The inner walls and bottom of the box were cleaned to prevent residual information from the previous animal (such as feces, urine, and odor) from affecting the results of subsequent tests. The video was digitally analyzed using EthoVision software. The animal was then replaced, and the experiment continued.

[0063] New Object Recognition: During the habituation phase, a test mouse was placed in the arena and allowed to explore for 2 minutes. After habituation, two objects of similar size but different shape and color were placed in opposite corners of the box, 5 cm from the side wall. The test mouse was then placed in the center of the arena and allowed to explore the arena containing both objects for 5 minutes. After 3 hours, one object was replaced by a new object similar in size to the previous object but different in shape and color. The same test mouse was then placed in the center and allowed to explore the arena and the two objects for 5 minutes. The exploration time and number of times the mouse explored the new and old objects were recorded. Recognition Index = New Object Exploration Time / (New Object Exploration Time + Familiar Object Exploration Time) × 100%; Discrimination Index = (New Object Exploration Time - Familiar Object Exploration Time) / (New Object Exploration Time + Familiar Object Exploration Time) × 100%.

[0064] Y-maze: Part 1 is the training phase. A partition is used to close the novel arm, and mice are placed in the remaining two arms and allowed to move freely for 5 minutes. Part 2 is conducted 5 hours later. Part 2 is the testing phase. The partition is removed, and mice are randomly placed in the arms and allowed to move freely for 5 minutes. The total number of times a mouse enters an arm and the number of times it enters the new arm are recorded within 5 minutes.

[0065] The results showed that ( Figure 4 In the open field experiment, compared with the normal control group (total movement distance 43809 mm; average movement speed 73.03 mm / s), sleep deprivation (total movement distance 64101 mm; average movement speed 109.3 mm / s) increased the total movement distance of mice by 46% (P<0.001) and the average movement speed by 50% (P<0.0001), indicating that sleep deprivation caused hyperactivity in mice. CCFM1320 intervention (total movement distance 50845 mm; average movement speed 87.48 mm / s) reduced the total movement distance of mice by 19% (P<0.05) and the average movement speed by 20% (P<0.01) compared with the model group, restoring them to near-normal levels and alleviating their hyperactivity. The control group (total movement distance 54786 mm; average movement speed 98.47 mm / s) reduced the total movement distance of mice by 14% compared to the model group (P=0.1544) and reduced the average movement speed of mice by 9% compared to the model group (P=0.2544), with no significant difference in either indicator. The CCFM1320 group reduced the total movement distance by 9% and the average movement speed by 12% compared to the control group.

[0066] In the novel object recognition experiment, compared with the control group (recognition index 0.5381; discrimination index 0.07624), sleep deprivation (recognition index 0.2643; discrimination index -0.4714) reduced the recognition index of mice by 50% (P<0.0001) and the discrimination index by 7 times (P<0.0001). This indicates that sleep deprivation reduces the mice's curiosity and willingness to explore new objects, significantly weakens their cognitive and learning abilities, and prevents them from retaining memories of old objects, thus making it impossible for them to distinguish between new and old objects. Their recognition and discrimination abilities are impaired. CCFM1320 intervention (recognition index 0.4139; discrimination index -0.1721) increased the recognition index of mice by 57% (P<0.05) and the discrimination index by 63% (P<0.05) compared to the model group, restoring them to near-normal levels and alleviating their impaired learning and memory abilities. The control bacteria intervention group (recognition index 0.2132; discrimination index -0.2993) showed a 19% decrease in the mouse recognition index compared to the model group, indicating a worse effect, while the discrimination index increased by 36% compared to the model group. Neither of these two indicators showed significant differences. The CCFM1320 group showed a 94% increase in the recognition index and a 47% increase in the discrimination index compared to the control bacteria group.

[0067] In the Y-maze experiment, compared to the control group (0.4225), sleep deprivation (0.2817) reduced the proportion of mice exploring new arms by 33% (P<0.01), indicating that sleep deprivation affects mice's ability to explore new environments, thus impairing their exploration ability. CCFM1320 intervention (0.3878) increased the proportion of mice exploring new arms by 38% compared to the model group (P<0.05), restoring them to near-normal levels and alleviating their impaired exploration ability. The control bacterial intervention group (0.265) reduced the proportion of mice exploring new arms by 6% compared to the model group, showing a worse effect. The CCFM1320 group increased the proportion of mice exploring new arms by 46% compared to the control bacterial group. Behavioral experiments have shown that CCFM1320 can alleviate sleep deprivation-induced hyperactivity and protect the exploration and memory functions of mice, while the control bacterial intervention has no effect on the behavior of mice.

[0068] Example 6: Lactobacillus helveticus CCFM1320 alleviates abnormal expression of circadian rhythm-related genes caused by sleep deprivation. An animal model was established according to the method in Example 3, and the hypothalamus was obtained according to the method in Example 4, and then subjected to real-time quantitative PCR. Table 3 Primers

[0069] Per1 , Per2 Circadian rhythm genes, as an important component of the circadian rhythm system, play a crucial role in the formation and regulation of circadian rhythms. Related research has demonstrated that... Per1 Mice with mutations and loss of expression lost the ability to regulate light-induced physiological rhythms, had shorter circadian rhythm cycles, and were affected by the expression of other clock genes. Per2 Gene mutations can lead to a shortened circadian rhythm, and abnormal changes in their expression and rhythm are closely related to the occurrence and development of cancer. Bmal1 These genes are two types of excitatory regulatory genes located in the suprachiasmatic nucleus. Increased expression of these genes leads to enhanced neural excitability, resulting in decreased sleep quality and reduced sleep duration. The results indicate that (…). Figure 5 ), compared to the normal control group ( Per1 The relative expression level of mRNA was 1.234; Per2 The relative expression level of mRNA was 0.9497; Bmal1 The relative expression level of mRNA was 0.8098, and sleep deprivation ( Per1 The relative expression level of mRNA was 0.7384; Per2 The relative expression level of mRNA was 1.745; Bmal1 The relative expression level of mRNA was 1.358, leading to hypothalamus Per1 A 40% decrease in relative gene expression (P<0.001) led to hypothalamic... Per2 The relative expression level of the gene increased by 84% (P<0.01), leading to hypothalamus Bmal1 The relative expression level of genes increased by 68% (P<0.0001). This indicates that sleep deprivation leads to abnormal expression of genes related to circadian rhythms in mice, resulting in impaired circadian rhythms. CCFM1320 intervention ( Per1 The relative expression level of mRNA was 1.033; Per2 The relative expression level of mRNA was 1.187; Bmal1 A relative mRNA expression level of 0.9788 can affect the hypothalamus of mice. Per1 The relative expression level of the gene was increased by 40% compared with the model group (P<0.05); it caused a 40% increase in the hypothalamus of mice. Per2 The relative expression level of the gene was reduced by 32% compared with the model group (P<0.05); it caused hypothalamus in mice to be affected. Bmal1 The relative gene expression level was reduced by 28% compared to the model group (P<0.01), returning to a near-normal level and helping mice restore their normal circadian rhythm. In contrast, the control bacterial intervention group ( Per1 The relative expression level of mRNA was 0.7854; Per2 The relative expression level of mRNA was 1.505; Bmal1 The relative expression level of mRNA was 1.448, which affected the hypothalamus of mice. Per1The relative expression level of mRNA increased by 6% compared with the model group (P=0.9666). Per2 The relative expression level of mRNA was 13% lower than that of the model group (P=0.5965). Bmal1 The relative expression level of mRNA increased by 6% compared with the model group (P=0.8405), and there were no significant differences among the three groups. CCFM1320 group Per1 The relative expression level of mRNA was 37% higher than that of the control group. Per2 The relative expression level of mRNA was 26% lower than that of the control bacteria intervention group. Bmal1 The relative expression level of mRNA was 32% lower than that of the control group.

[0070] The results showed that CCFM1320 intervention could alleviate the abnormal expression of circadian rhythm-related genes caused by sleep deprivation and regulate the circadian rhythm in mice, while the control bacterial intervention group had no effect on the expression of hypothalamic circadian rhythm-related genes.

[0071] Example 7: Using the Lactobacillus helveticus CCFM1320 of this invention to produce fermented food containing this bacterium. Select fresh vegetables (such as cucumbers, carrots, beets, celery, cabbage products, or a mixture of several), wash them, juice them, and then perform high-temperature instantaneous sterilization. After sterilization at 140 ℃ for 2 seconds, immediately cool down to 37 ℃, and then inoculate with a bacterial agent or fermentation agent containing Lactobacillus helveticus CCFM1320, so that the concentration of Lactobacillus helveticus CCFM1320 after inoculation reaches 1×10⁻⁶. 8 Fruit and vegetable beverages containing live Lactobacillus helveticus CCFM1320 were obtained by storing the beverage at a temperature of 4°C with a concentration of CFU / mL or higher. Other fermented foods are prepared using Lactobacillus helveticus CCFM1320 fermentation, including solid foods, liquid foods, and semi-solid foods. These fermented foods include dairy products, soy products, and fruit and vegetable products, with dairy products including milk, sour cream, and cheese. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Lactobacillus helveticus ( Lactobacillus helveticus CCFM1320 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC NO: 63716.

2. A microbial preparation containing the Lactobacillus helveticus CCFM1320 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The amount of *Lactobacillus helveticus* CCFM1320 added shall not be less than 5 × 10⁻⁶. 9 CFU / g or 5×10 9 CFU / mL.

4. The use of Lactobacillus helveticus CCFM1320 as described in claim 1 or any of the microbial preparations described in claims 2 to 3 in the preparation of a drug for sleep deprivation-induced circadian rhythm disorders.

5. The application according to claim 4, characterized in that, The drug contains the Lactobacillus helveticus CCFM1320, as well as a drug carrier and / or pharmaceutical excipients.

6. A drug for regulating circadian rhythm disorders caused by sleep deprivation, characterized in that, Contains Lactobacillus helveticus CCFM1320 as described in claim 1.

7. The use of Lactobacillus helveticus CCFM1320 as described in claim 1 in the preparation of health products that help improve sleep.

8. The application of Lactobacillus helveticus CCFM1320 as described in claim 1 in the preparation of food.

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