Weizwinia coagulans CN365 for relieving anxiety and assisting sleep and application of Weizwinia coagulans CN365

Abstract

The invention provides an anxiety relieving and sleep assisting strain of Weizia coagulans and application of the anxiety relieving and sleep assisting strain of Weizia coagulans, the Weizia coagulans is Weizia coagulans CN365, the Weizia coagulans is preserved in China Center for Type Culture Collection, the preservation date is October 28, 2025, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 20252374. The invention provides a brand-new strain of Witzia ciliaris CN365, which has the effects of relieving anxiety and assisting sleep. Experimental results show that the bacillus coagulans CN365 is resistant to high temperature and high in yield of indole lactic acid, and particularly suitable prebiotics are added, so that high yield of indole lactic acid can be remarkably promoted. Freeze-dried powder is prepared, and the activity of the bacterial powder is high; besides, the Witzia ciliaris CN365 can improve anxiety, improve sleep disorders and promote calcium transport and absorption, and especially when the Witzia ciliaris CN365 and the Lactobacillus crispatus GG77 are combined for use, the Witzia ciliaris CN365 and the Lactobacillus crispatus GG77 have a synergistic interaction effect, and the transport and absorption effect on calcium ions can be significantly improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Weizmannia coagulans, and particularly relates to Weizmannia coagulans CN365 for relieving anxiety and assisting sleep and application thereof. BACKGROUND

[0002] With the acceleration of social rhythm, more and more people begin to have the sub-health problems of increased anxiety and sleep disorders. Anxiety is the psychosomatic reaction of an individual when facing external challenges, internal needs or environmental changes due to perceived threats or imbalance. It can be triggered by specific events (such as work, interpersonal relationships) or long-term states (such as economic burden, health problems), and is manifested as emotional fluctuations, physiological changes or behavioral abnormalities. Moderate stress can stimulate motivation, but long-term or excessive stress or anxiety can harm health. Anxiety is the stress response of the brain to "threats" or "challenges". When an individual believes that their ability or resources are insufficient to cope with external demands, stress arises. Common sources include: external events: such as work deadlines, family conflicts, economic difficulties, etc.; 1, internal conflicts: such as the gap between ideals and reality, value conflicts; 2, chronic stressors: long-term exposure to noise, illness, etc. Sleep disorders refer to abnormalities in sleep quality, timing, rhythm or behavior that affect physical and mental health. It not only includes common insomnia, but also covers various types such as sleepiness, sleep breathing disorders, parasomnias, etc. Common manifestations of sleep disorders include: 1, insomnia: difficulty falling asleep, light sleep, easy wakefulness, early awakening, etc.; 2, poor sleep quality: feeling tired and weak after sleep; 3, sleep sensation disorder: lack of sleep real sense, but confirmed by others that they have fallen asleep; 4, impaired daytime functioning: decreased memory, emotional abnormalities (such as anxiety and irritability), daytime sleepiness, etc.

[0003] Currently, in addition to self-regulation and reducing external stress, severe problems such as anxiety and sleep disorders may require drug intervention, but current drugs are usually chemical drugs, which have the defects of drug dependence and side effects. SUMMARY

[0004] The application provides Weizmannia coagulans CN365 for relieving anxiety and assisting sleep and application thereof, aiming at the problems in the prior art.

[0005] Technical scheme: In order to achieve the above application purposes, the application adopts the following technical scheme: In a first aspect, the present invention provides a strain of Weizmannia coagulans that relieves anxiety and aids sleep, wherein the Weizmannia coagulans strain is Weizmannia coagulans CN365, deposited at the China Center for Type Culture Collection on October 28, 2025, with accession number CCTCC NO: M 20252374.

[0006] In a second aspect, the present invention provides a composition comprising the aforementioned Weizmann's coagulans CN365.

[0007] As one embodiment, the composition is a lyophilized powder containing *Weizmannii coagulans* CN365; preferably, the activity of *Weizmannii coagulans* CN365 in the lyophilized powder is 1.0 × 10⁻⁶. 10 ~1.0×10 12 CFU / g.

[0008] Thirdly, the present invention provides a culture medium for culturing *Weizmannii coagulans* CN365, the culture medium comprising the following components: Soybean peptone 8.0-20.0 g / L, yeast extract 10.0-20.0 g / L, anhydrous glucose 15.0-20.0 g / L Sucrose 5.0-10.0 g / L, dipotassium hydrogen phosphate 1.0-2.5 g / L Diamine hydrogen citrate 1.0-2.5 g / L, sodium acetate 4.0-6.0 g / L, magnesium sulfate 0.05-0.2 g / L. Manganese sulfate 0.4-0.6 g / L, L-cysteine ​​salt 0.5-1.5 g / L, Tween 80 0.5-1.5 ml / L; In addition, it also includes 1.0-2.5 g / L of calcium carbonate and / or 2.5-5.0 g / L of soybean lecithin, preferably a combination of calcium carbonate and soybean lecithin.

[0009] More preferably, the culture medium comprises the following components: Soybean peptone 18.5 g / L, yeast extract 10.0 g / L, anhydrous glucose 15.0 g / L, sucrose 8.5 g / L, dipotassium hydrogen phosphate 2.0 g / L, diamine hydrogen citrate 1.5 g / L, sodium acetate 5 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.5 g / L, calcium carbonate 1.8 g / L, L-cysteine ​​salt 1 g / L, Tween 80 1 ml / L, soybean lecithin 2.5 g / L.

[0010] Fourthly, the present invention provides a method for culturing the aforementioned Weizmann's coagulans CN365, comprising inoculating Weizmann's coagulans CN365 at an inoculation rate of 3%-10% using the above-mentioned culture medium, and carrying out fermentation culture at 35℃-37℃ for 8-16 hours; preferably, the inoculation rate is 8%.

[0011] Fifthly, the present invention provides the use of the aforementioned Weizmannii coagulans CN365 and the above-described composition in the preparation of products that improve anxiety, improve sleep disorders, promote calcium transport and absorption, and / or improve acne.

[0012] In a sixth aspect, the present invention provides the application of the aforementioned Weizmann's coagulans CN365 in the production of indole-lactic acid.

[0013] As a specific implementation plan, the application includes adding 1.0-2.0 g / L tryptophan and 1.0-3.0 g / L inulin to the culture medium, inoculating with Weizmannia coagulans CN365, and then culturing at 35℃-37℃ for 40-60 h; Preferably, the culture medium comprises the following components: Soybean peptone 8.0-20.0 g / L, yeast extract 10.0-20.0 g / L, anhydrous glucose 15.0-20.0 g / L Sucrose 5.0-10.0 g / L, dipotassium hydrogen phosphate 1.0-2.5 g / L Diamine hydrogen citrate 1.0-2.5 g / L, sodium acetate 4.0-6.0 g / L, magnesium sulfate 0.05-0.2 g / L. Manganese sulfate 0.4-0.6 g / L, L-cysteine ​​salt 0.5-1.5 g / L, Tween 80 0.5-1.5 ml / L. Calcium carbonate 1.0-2.5 g / L, soybean lecithin 2.5-5.0 g / L.

[0014] In a seventh aspect, the present invention provides a composition comprising the aforementioned *Weizmannii coagulans* CN365 and *Lactobacillus crispatus* GG77, wherein *Lactobacillus crispatus* GG77 is deposited at the China Center for Type Culture Collection (CCTCC) on November 21, 2025, with accession number CCTCC NO: M 20252646.

[0015] Eighthly, the present invention also provides the use of the above-described composition of Weizmannii coagulans CN365 and Lactobacillus curvature GG77 in the preparation of products that promote calcium transport and absorption.

[0016] Beneficial Effects: Compared with existing technologies, this invention provides a novel strain of *Weizmannii coagulans* CN365, which has the effects of relieving anxiety and aiding sleep. Experimental results show that *Weizmannii coagulans* CN365 is heat-resistant and produces high levels of indole-lactate, especially with the addition of suitable prebiotics, which can significantly promote high production of indole-lactate. When prepared as a freeze-dried powder, the bacterial powder has high activity. Furthermore, *Weizmannii coagulans* CN365 can improve anxiety, improve sleep disorders, and promote calcium transport and absorption. In particular, when *Weizmannii coagulans* CN365 is used in combination with *Lactobacillus curvatureii* GG77, the two have a synergistic effect, which can significantly improve the transport and absorption of calcium ions. Attached Figure Description

[0017] Figure 1 Gram staining for Weizmann's coagulans strain CN365.

[0018] Figure 2 Evolutionary tree of Weizmannii strain CN365.

[0019] Figure 3 The effect of different groups of probiotics on the intracellular calcium ion concentration of Caco-2 cells. Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0021] Example 1: Preliminary screening of highly active culturable strains To further obtain beneficial bacterial strains for human health and to supplement the human gut microbiota, target strains were obtained through screening of the human gut microbiota. Healthy gut samples were collected from young adults with good physical indicators but a BMI ≤15. These samples were serially diluted and spread onto MRS agar medium. The same sample was spread twice, and cultured in anaerobic and 5% oxygen environments respectively, both at 35-37℃. Single colonies were selected from the MRS agar medium and placed in 15ml of MRS liquid medium. The selected single colonies were then incubated on a shaker at 35-37℃ for 8-10 hours to obtain the fermentation broth. Gram staining and OD detection of the fermentation broth were then performed. 600 The viable cell count in the fermentation broth was observed under a microscope. Gram-negative samples, samples with poor liquid fermentation results, or samples without obvious bacterial proliferation were discarded. A total of 152 colony fermentation samples were obtained. 0.5 ml of each of the remaining fermentation broths was used for bacterial sequencing and identification, and 2 ml of each was added to 2 ml of 30%-40% glycerol solution, pre-cooled, and frozen at -80°C for preservation as candidate strains. Thus, a preliminary Gram-positive human bacterial strain capable of being cultured under laboratory conditions was obtained.

[0022] Example 2 Screening of highly thermostable strains To screen for strains capable of withstanding high temperatures, the following steps were performed: Step 1: 50 μl of each of the 152 strains screened in Example 1 was inoculated into 5 ml of MRS liquid medium and cultured on a shaker at 35-37°C for 8-10 h; Step 2: The fermentation broth was placed in a 65°C incubator for 4 h; Step 3: 10 ml of MRS medium was added to each culture tube that had undergone high-temperature incubation in Step 2, and the tubes were cultured on a shaker at 35-37°C for 6 h; Step 4: The OD of the fermentation broth was initially measured. 600 The viable cell count in the fermentation broth was observed under a microscope. Samples with good viable cell counts from step three were retained, while most samples without viable cells were discarded. Step five: The viable cell count in the remaining fermentation broth samples from step four was accurately determined using the plate coating method. Strains that remained active after high-temperature incubation were selected, resulting in three remaining strains, numbered CN05, CN365, and CN2406. If these strains are further evaluated to be beneficial to humans, they possess heat-resistant properties, facilitating the preparation of microbial formulations for room-temperature storage and long-term stability.

[0023] Example 3 Screening of indole-producing strains To obtain a strain with high activity, heat resistance, and high indolelacic acid production, *Lactobacillus rhamnosus* GG strain was used as a control strain. The three strains screened in Example 2 were activated, diluted to a uniform cell concentration, and inoculated into 50 ml MRS medium at a volume ratio of 5%, and cultured at 37°C for 48 h. The indolelacic acid concentration in the fermentation broth was detected by HPLC-MS / MS, and the results are shown in Table 1. The results indicate that the indolelacic acid production in the fermentation broth of strain CN365 was significantly higher than that of other strains, making it a target strain for high indolelacic acid production.

[0024] Table 1. Indolelactic acid yield in fermentation broth of each strain

[0025] Example 4: Identification of strains and archiving of strains Based on the evaluation results during the comprehensive screening process, strain CN365, which exhibits high activity, high temperature resistance, and high indolelactate production, was selected as the target strain.

[0026] (1) Morphological identification Observation of the purified bacterial solution under a microscope revealed that the cells were rod-shaped, with some cells having oval microspores at one end. The cells were rod-shaped, without branching or flagella. The colonies of the strain on MRS agar medium were grayish-white, semi-circular, raised, with a smooth, glossy surface and neat edges.

[0027] (2) Gram staining Gram staining result is Gram positive, such as Figure 1As shown.

[0028] (3) Molecular biological identification of 16S rRNA The 16S rDNA gene sequence of strain CN365 was amplified and sequenced using published universal 16S primers (primer sequences: 8F: 5'-AGAGTTTGATCCTGGCTCA-3'; 1510R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing identification. The nucleotide sequence of the 16S rDNA of strain CN365 is sequence 1 in the sequence listing. After 16S rDNA gene alignment, the similarity rate with Weizmannia coagulans in Genebank reached 99%. Combined with microbial system identification, strain CN365 was identified as a Weizmannia coagulans strain, named Weizmannia coagulans CN365. Its 16S rDNA is shown in SEQ ID NO.1, and the phylogenetic tree is shown below. Figure 2 As shown.

[0029] Weizmannia coagulans CN365, a strain identified by biological analysis, was deposited at the China Center for Type Culture Collection (CCTCC) on October 28, 2025, with accession number CCTCC NO: M 20252374, at Wuhan University, Wuhan, Hubei Province, People's Republic of China.

[0030] Example 5: Industrial fermentation and freeze-dried powder preparation of *Weizmannii coagulans* CN365 To facilitate industrial-scale cultivation, fermentation tests on culture medium and preparation of freeze-dried powder were conducted on Weizmann's coagulans CN365.

[0031] (1) Culture medium preparation.

[0032] The CN365 medium for *Weizmannii coagulans* was prepared using the following formula: 18.5 g / L soybean peptone, 10.0 g / L yeast extract, 15.0 g / L anhydrous glucose, 8.5 g / L sucrose, 2.0 g / L dipotassium hydrogen phosphate, 1.5 g / L diammonium citrate, 5 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 1.8 g / L calcium carbonate, 1 g / L L-cysteine ​​sulfate, 1 ml / L Tween 80, and 2.5 g / L soybean lecithin. All raw materials were food-grade.

[0033] The preparation scheme 2 for the CN365 medium of *Weizmannia coagulans* was as follows: 8.0 g / L soybean peptone, 8.0 g / L yeast extract, 12.0 g / L anhydrous glucose, 5.0 g / L sucrose, 1.0 g / L dipotassium hydrogen phosphate, 1.0 g / L diammonium citrate, 5 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 1.8 g / L calcium carbonate, 1 g / L L-cysteine ​​sulfate, and 1 ml / L Tween 80. All raw materials were food-grade.

[0034] The preparation scheme 3 for the CN365 medium of *Weizmannia coli* is as follows: 20.0 g / L soybean peptone, 20.0 g / L yeast extract, 20.0 g / L anhydrous glucose, 10.0 g / L sucrose, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L diammonium citrate, 5 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 1 g / L L-cysteine ​​sulfate, 1 ml / L Tween 80, and 5 g / L soybean lecithin. All raw materials are food-grade.

[0035] (2) Fermentation culture After preparing the culture medium, adjust the pH to 6.5, sterilize at 115℃ for 30 min, and inoculate it into the fermenter at an inoculation rate of 3%-10%, preferably 8%. Incubate continuously at 35℃-37℃ for 8-16 hours. At 12 h, the viable cell count of the fermentation broth under the three culture medium schemes was measured to be 2.5 × 10⁻⁶. 8 CFU / mL, 1.7 × 10 8 CFU / mL, 2.2 × 10 8 The viable cell count of the fermentation broth was measured at CFU / mL at 16 h to be 3.3 × 10⁻⁶. 9 CFU / mL, 1.8 × 10 9 CFU / mL, 2.1×10 9 The CFU / mL viable cell count is already high, meeting the requirements for industrial-scale high-density fermentation production. The preferred culture medium configuration scheme 1 has advantages in terms of fermentation cell growth rate and production ratio, and is therefore the preferred culture medium scheme for Lactobacillus rhamnosus.

[0036] (3) Centrifugation and freeze drying After 12 hours of cultivation, once the growth curve reached a plateau based on the OD600 value, the fermentation broth was rapidly cooled to below 15°C and centrifuged at 5000 rpm using a tubular centrifuge to harvest the mycelium sludge. The harvested mycelium sludge was diluted to a moisture content of 60%-80% and emulsified with an equal weight of freeze-drying protectant. The protectant consisted of: 36% skim milk powder, 15% stachyose, 15% mannitol, 3% Tween-80, 15% betaine, 1% monosodium glutamate, and 15% soluble starch. The emulsified mycelium sludge was then freeze-dried in a freeze dryer until the final material had a moisture content of less than 5% and a water activity of 0.11-0.22 aw.

[0037] The freeze-dried bacterial powder has an activity level of up to 1.2 × 10⁻⁶. 11 The CFU / g concentration facilitates storage and makes it suitable for use as a food additive (lactobacco powder) or in the preparation of probiotic formulations.

[0038] Example 6: Prebiotic combination that promotes high indole-lactic acid production by *Weizmannii coagulans* CN365 To promote high indole-lactic acid production by *Weizmannii coagulans* CN365 during use, the culture medium scheme 1 in Example 5 was optimized. Scheme a: 1.5 g / L tryptophan was added to culture medium scheme 1; Scheme b: 2.0 g / L inulin was added to culture medium scheme 1; Scheme c: 1.5 g / L tryptophan and 2.0 g / L inulin were added to culture medium scheme 1; Scheme d: 2.0 g / L galactooligosaccharides were added to culture medium scheme 1; Scheme e: 1.5 g / L arginine was added to culture medium scheme 1; Scheme f: 1.5 g / L arginine and 2.0 g / L inulin were added to culture medium scheme 1. According to the different culture medium schemes, culture medium scheme 1 was used as the control medium, and each culture was inoculated into 50 ml of MRS medium at a volume ratio of 5% and cultured at 37°C for 48 h. The concentration of indole-lactic acid in the fermentation broth was detected by HPLC-MS / MS, and the results are shown in Table 2. The results showed that the indole-lactate yield in the fermentation broth of *Weizmannia coagulans* CN365 fermented in medium C was significantly higher than that in other mediums and media. This indicates that not all amino acids and prebiotics can promote high indole-lactate production in *Weizmannia coagulans* CN365. Furthermore, the combination of tryptophan and inulin can promote high indole-lactate production in *Weizmannia coagulans* CN365. In practical applications, tryptophan and inulin can be used as excipients to improve the indole-lactate metabolic yield of *Weizmannia coagulans* CN365.

[0039] Table 2. Indolelactic acid yield of *Weizmannii* CN365 fermentation medium in various media.

[0040] Example 7: The use of *Weizmannii coagulans* CN365 to improve anxiety symptoms The gut-brain axis plays a crucial role in maintaining gut homeostasis and brain function through bidirectional communication. Anxiety symptoms are closely related to the levels of prostaglandins and neurotransmitters in the brain. To test the effect of *Weizmannii coagulans* CN365 on anxiety via the gut-brain axis, 40 subjects aged 20-45 years who were not using medication and scored over 60 on the Self-Assessment Depression Scale (SDS) were randomly assigned to either a control group (n=20) or an observation group (n=20). The observation group received lactic acid bacteria prepared with *Weizmannii coagulans* CN365 (2g / sachet, containing 1.5 × 10⁻⁶ *Weizmannii coagulans* CN365). 10 CFU / sachet (with maltodextrin as the excipient), the control group received the same dose of maltodextrin (2g / sachet, maltodextrin), one sachet twice daily, morning and evening. Participants were instructed to avoid fermented milk and other foods containing live lactic acid bacteria, or other products containing probiotics or prebiotics. The intervention lasted approximately 30 days. After the intervention began, the Self-Rating Depression Scale (SDS) questionnaire was administered every 7 days to assess mood symptoms.

[0041] Among the anxiety symptoms listed on the Self-Rating Depression Scale (SDS), the CN365 observation group scored significantly lower than the placebo group in "depressed mood," "anxiety," "tension," "orientation," and "memory." The data indicate that daily intake of CN365 can improve symptoms such as anxiety.

[0042] Example 8: *Weizmannii coagulans* CN365 improves sleep disorders Sleep is generally divided into four stages: falling asleep, light sleep, deep sleep, and prolonged deep sleep. The sleep monitoring function of the Xiaomi Mi Band (model M2434B1) is used to statistically evaluate sleep quality. The Mi Band monitors sleep based on four indicators: deep sleep, light sleep, REM (rapid eye movement sleep, also known as REM sleep, is a deeper stage of sleep), and wakefulness, and assigns a sleep score. Sleep disorder intervention tests are conducted on individuals meeting the following criteria: age 20-60 years; Pittsburgh Sleep Quality Index (PSQI) score ≥ 11; normal blood pressure: systolic blood pressure 90-140 mmHg, diastolic blood pressure 60-90 mmHg; regular sleep schedule; no consumption of probiotic products (excluding yogurt, lactic acid bacteria drinks, etc.) within the past two weeks; no use of antibiotics within the past month; and no use of psychiatric medications or sleeping pills within the past two months. Twenty-four eligible participants were randomly divided into two groups: a control group (n=12) and an observation group (n=12). The observation group received lactic acid bacteria (2g / bag, containing 2.0×10⁻⁶ Wickeman's coagulans CN365) prepared from *Wickeman's coagulans* CN365. 10The control group received the same dose of maltodextrin (2g / sachet, maltodextrin as the main ingredient), one sachet twice daily, morning and evening, for approximately 28 days. The results showed that after 28 days of intervention, the Pittsburgh Sleep Quality Index (PSI) in the observation group significantly improved, with an average score below 10. Participants experienced a reduction in sleep disturbances from moderate to severe to mild to moderate. The observation group also saw an increase in both deep sleep time and total sleep duration. This indicates that *Weizmannii coagulans* CN365 has a function in improving sleep disorders.

[0043] Table 3 Statistical Table of Improvement in Sleep Disorders

[0044] Example 9: Promoting Calcium Transport and Absorption To test the effect of *Weizmannii coagulans* CN365 on calcium ion transport and absorption in intestinal mucosal cells, the strain was mixed with fully differentiated human colon adenocarcinoma Caco-2 cells at a ratio of 2 × 10⁻⁶. 4 Cells were cultured per well in black 96-well plates. After 24 hours of adherent cell culture, different experimental bacterial strains were added and incubated with the adherent cells for another 24 hours. Calcium chloride was then injected, followed by the addition of prepared Flow-4-AM solution, and the plates were incubated for 30 minutes. Once the fluorescent dye entered the cells, it was digested by esterases and reacted with intracellular calcium... 2+ The combination produces a strong fluorescence effect. The calcium uptake of Caco-2 cells was detected by using the Flow-4-AM fluorescent probe at an excitation wavelength of 490 nm and an emission wavelength of 525 nm.

[0045] The experimental group was divided into two groups: T1 (20 μL of coagulated Weizmannii CN365 bacterial suspension, containing approximately 2.0 × 10⁻⁶ viable cells). 6 CFU), T2 group (20 μL Lactobacillus curlifron GG77 bacterial suspension, containing approximately 2.0 × 10⁻⁶ live bacteria). 6 CFU), Group T3 (20 μL of mixed bacterial culture of Lactobacillus curlis GG77 and Weizmannii coagulans CN365, GG77:CN365 ratio 1:1, total viable count approximately 2.0 × 10⁻⁶ CFU), T3 group (20 μL of mixed bacterial culture of Lactobacillus curlis GG77 and Weizmannii coagulans CN365, GG77:CN365 6 CFU), Group T4 (20 μL of mixed bacterial suspension of Lactobacillus curlis GG77 and Weizmannii coagulans CN365, GG77:CN365 ratio 1:3, total viable count approximately 2.0 × 10⁻⁶ CFU). 6 CFU), Group T5 (20 μL of mixed bacterial culture of Lactobacillus curlis GG77 and Weizmannii coagulans CN365, GG77:CN365 ratio 3:1, total viable count approximately 2.0 × 10⁻⁶ CFU). 6CFU), with the blank control group being the BL group (20 μL of fermentation supernatant without viable bacteria). Lactobacillus crispatus GG77 used in the experiment was tested as a co-culture (the Lactobacillus crispatus GG77 used is deposited at the China Center for Type Culture Collection (CCTCC) on November 21, 2025, accession number CCTCC NO: M 20252646, deposit address: Wuhan University, Wuhan, Hubei Province, People's Republic of China).

[0046] By measuring intracellular Ca in adherent Caco-2 cells 2+ The fluorescence intensity was used to assess the effects of different groups of probiotics and CaCl2 concentrations on intracellular CaCO-2 cells. 2+ The effect of content. For example Figure 3 As shown, in the same Ca 2+ At what concentration, intracellular Ca in cells cultured in each experimental group 2+ The fluorescence intensity of the T3 group was significantly higher than that of the control group; compared with other groups, the T3 group (GG77:CN365 = 1:1) showed significantly higher fluorescence intensity at different Ca2+ levels. 2+ Intracellular Ca under mass concentration conditions 2+ The fluorescence intensity of these groups was significantly higher than that of other groups. These results indicate that *Weizmannii coagulating* CN365 and / or *Lactobacillus curvature* GG77 can promote Ca2+ coagulation. 2+ Entering adherent Caco-2 cells, thereby allowing intracellular Ca2+ to enter the cells. 2+ Increased levels can promote the transport and absorption of calcium ions by the intestinal mucosa; a 1:1 combination of *Weizmannii coagulans* CN365 and *Lactobacillus curvatureii* GG77 promotes the absorption of calcium ions. 2+ The effect is better when it enters adherent cells, that is, the combination of CN365 and GG77 in a 1:1 ratio promotes the transport and absorption of calcium ions in the intestinal mucosa.

[0047] Example 10: Interventional effect of *Weizmannii coagulans* CN365 on acne. Based on the sleep-aiding effect and high indole-lactate production of *Weizmannii coagulans* CN365, 86 participants meeting the following criteria were selected: 1) Age 12-35 years, gender not limited; 2) Meeting the diagnostic criteria for mild to moderate acne vulgaris in the *Chinese Guidelines for Acne Treatment (2023 Revised Edition)*: lesions mainly consisting of comedones and inflammatory papules, number <50, inflammatory nodules <3, and a GAGS (General Acne Grading System) score of 5-18; 3) Not having used any topical or oral acne medications in the past 2 weeks; 4) Willing to participate in this study and able to cooperate in completing the entire experimental period and follow-up. Patients were randomly divided into an intervention group (T) and a control group (B), with 43 patients in each group. The intervention group received *Weizmannii coagulans* CN365 lyophilized powder (2g per sachet, containing 2.0 × 10⁻⁶ *Weizmannii coagulans* CN365) that met food safety requirements. 10 The control group received a placebo (2g maltodextrin per packet) that met food safety requirements, dissolved in warm water, one packet each time, twice a day for 28 consecutive days. The improvement of acne in the intervention and control groups was assessed. Changes in the General Acne Grading System (GAGS) scores before and after intervention were statistically analyzed. GAGS scores were graded based on the number and severity of lesions (comedones, inflammatory papules, nodules) in six facial regions (forehead, right cheek, left cheek, nose, jaw, and chest / back; lesions on the chest / back can be ignored if there are few lesions). Each region was scored from 0 to 4 points based on severity, and the total score was the sum of the scores for each region. A higher score indicated more severe acne. The difference in GAGS scores before and after intervention and the rate of decrease were calculated. The efficacy criteria were: cured (GAGS score decrease rate ≥ 90%), significantly effective (60% ≤ decrease rate < 90%), effective (20% ≤ decrease rate < 60%), and ineffective (decrease rate < 20%). The overall effective rate was calculated as (number of cured cases + number of cases with significant improvement + number of cases with improvement) / total number of cases × 100%. The results are shown in Table 4. According to the statistical results, the intervention group showed significant improvement in acne symptoms, and the overall effective rate was significantly higher than that of the control group. This indicates that oral supplementation with *Weizmannii coagulans* CN365 can effectively intervene in acne symptoms and can be used in the preparation of related pharmaceutical products.

[0048] Table 4. Intervention effect of *Weizmannii coagulans* CN365 on acne.

[0049] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A strain of *Weizmannii* that relieves anxiety and aids sleep, characterized in that, The *Weizmannia coagulans* strain mentioned is *Weizmannia coagulans* CN365, deposited at the China Center for Type Culture Collection (CCTCC) on October 28, 2025, with accession number CCTCC NO: M 20252374.

2. A composition, characterized in that, The composition comprises the *Weizmannii coagulans* CN365 as described in claim 1.

3. The composition according to claim 2, characterized in that, The composition is a lyophilized powder containing *Weizmannii coagulans* CN365; preferably, the activity of *Weizmannii coagulans* CN365 in the lyophilized powder is 1.0 × 10⁻⁶. 10 ~1.0×10 12 CFU / g.

4. A culture medium for culturing *Weizmannii coagulans* CN365, characterized in that, The culture medium comprises the following components: Soybean peptone 8.0-20.0 g / L, yeast extract 10.0-20.0 g / L, anhydrous glucose 15.0-20.0 g / L, sucrose 5.0-10.0 g / L, dipotassium hydrogen phosphate 1.0-2.5 g / L, diamine hydrogen citrate 1.0-2.5 g / L, sodium acetate 4.0-6.0 g / L, magnesium sulfate 0.05-0.2 g / L, manganese sulfate 0.4-0.6 g / L, L-cysteine ​​salt 0.5-1.5 g / L, Tween 80 0.5-1.5 ml / L; In addition, it also includes 1.0-2.5 g / L of calcium carbonate and / or 2.5-5.0 g / L of soybean lecithin, preferably a combination of calcium carbonate and soybean lecithin.

5. The method for culturing *Weizmannii coagulans* CN365 according to claim 1, characterized in that, The method includes using the culture medium described in claim 4, inoculating *Weizmannii coagulans* CN365 at an inoculation rate of 3%-10%, and fermenting the culture at 35℃-37℃ for 8-16 hours; preferably, the inoculation rate is 8%.

6. The use of the *Weizmannii coagulans* CN365 of claim 1, or the composition of claim 2 or 3, in the preparation of products that improve anxiety, improve sleep disorders, promote calcium transport and absorption, and / or improve acne.

7. The application of the *Weizmannii coagulans* CN365 as described in claim 1 in the production of indole-lactic acid.

8. The application according to claim 7, characterized in that, The application involves adding 1.0-2.0 g / L tryptophan and 1.0-3.0 g / L inulin to the culture medium, inoculating with *Weizmannia coagulans* CN365, and then culturing at 35℃-37℃ for 40-60 h. Preferably, the culture medium comprises the following components: Soybean peptone 8.0-20.0 g / L, yeast extract 10.0-20.0 g / L, anhydrous glucose 15.0-20.0 g / L Sucrose 5.0-10.0 g / L, dipotassium hydrogen phosphate 1.0-2.5 g / L, diamine hydrogen citrate 1.0-2.5 g / L, sodium acetate 4.0-6.0 g / L, magnesium sulfate 0.05-0.2 g / L, manganese sulfate 0.4-0.6 g / L, L-cysteine ​​salt 0.5-1.5 g / L, Tween 80 0.5-1.5 ml / L, calcium carbonate 1.0-2.5 g / L, soybean lecithin 2.5-5.0 g / L.

9. A composition, characterized in that, The composition comprises *Weizmannii coagulans* CN365 as described in claim 1 and *Lactobacillus crispatus* GG77, which is deposited at the China Center for Type Culture Collection (CCTCC) on November 21, 2025, with accession number CCTCC NO: M 20252646.

10. Use of the composition of claim 9 in the preparation of a product that promotes calcium transport and absorption.

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