Application of clostridium tomentosum in preparation of product for resisting body injury caused by staying up late

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses application of clostridium algiicola to preparation of a product for resisting body injury caused by staying up late. The invention also relates to an application of clostridium dalinum in preparation of a product for preventing, treating or improving body injury caused by staying up late. The invention discloses an application of clostridium mali in preparation of a product for resisting body injury caused by staying up late, which realizes prevention, improvement and treatment of body injury caused by staying up late through multi-target synergistic effect of repairing intestinal barrier, regulating flora balance, antagonizing oxidative stress and inflammation and improving metabolism and neuroprotection.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Clostridium daliense in the preparation of products that combat damage to the body caused by staying up late. Background Technology

[0002] Staying up late means a shortened total sleep time (insufficient sleep) and / or a decline in sleep quality due to the sleep onset time not conforming to the body's internal rhythm (such as reduced deep sleep and fragmented sleep). Based on the cause, staying up late is divided into two types: active and passive (such as insomnia, environmental interference, etc.). Due to multiple factors such as changes in lifestyle, increased entertainment options, increased work pressure, and fluctuations in psychological state, staying up late is prevalent in modern society and has become a widespread health threat. Statistics show that approximately 30% of shift workers and 15% of people with sleep disorders worldwide face high-risk exposure to staying up late. Staying up late exposes the body to systemic damage through a triple pathological cascade: First, it disrupts the circadian rhythm, interfering with the coupling of biological clock genes such as CLOCK / BMAL1 in the brain, leading to disordered synthesis of antioxidant enzymes mediated by the Nrf2 pathway, weakening the body's ability to scavenge free radicals, causing the accumulation of oxidative stress and inflammatory factors, and disrupting cellular homeostasis. Second, it causes an imbalance in the gut microbiota-metabolic axis, inhibiting the proliferation of beneficial bacteria and enriching pathogenic bacteria, leading to disordered synthesis of microbial metabolites (especially short-chain fatty acids), weakening the intestinal mucus barrier, and reducing Claurum levels. The expression of tight junction proteins such as din-1, ZO-1, and Occludin can also lead to systemic energy metabolism imbalance. Thirdly, the extravasation of inflammatory factors in the gut-brain axis can cause a large accumulation of ROS in the gut, exacerbating damage to the intestinal barrier and allowing endotoxins such as lipopolysaccharide (LPS) to enter the bloodstream. This activates systemic inflammatory factors, which activate TLR4 receptors through the vagus nerve, exacerbating the activation of microglia in the brain, increasing neuroinflammatory factors in the hippocampus, and causing nerve damage, metabolic disorders, and cardiovascular disease. Ultimately, this may lead to multiple organ failure and death.

[0003] Current interventions for the damage caused by staying up late have significant limitations: while commercially available probiotic preparations can upregulate intestinal tight junction proteins, regulate gut microbiota, and exert some antioxidant effects, their inhibitory effects on inflammatory factors are limited; traditional antioxidants can reduce levels of ROS and MDA in the intestine and serum, but they are difficult to repair intestinal structure and clear inflammatory factors in the gut-brain axis; commonly used clinical drugs, such as melatonin receptor agonists and benzodiazepines... Sedative-like drugs can only regulate sleep cycles in the short term and cannot block the leakage of inflammatory factors; furthermore, long-term use may disrupt neurocognitive function. Therefore, developing a comprehensive intervention that can simultaneously regulate gut microbiota homeostasis, repair gut barrier function, antagonize oxidative stress and inflammation, and regulate circadian rhythm disorders has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] This invention aims to provide the application of Clostridium daliense in the preparation of products for combating bodily damage caused by staying up late. Through the multi-target synergistic effects of repairing the intestinal barrier, regulating the balance of the gut microbiota, antagonizing oxidative stress and inflammation, improving metabolism and neuroprotection, it achieves the prevention, improvement and treatment of bodily damage caused by staying up late.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] Application of Clostridium dalinum in the preparation of products for the prevention, treatment or improvement of bodily damage caused by staying up late.

[0007] Preferably, the Clostridium dalinum is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 27789 and deposit date of July 3, 2023.

[0008] Preferably, the product comprises live cells, inactivated cells, metabolites and / or fermentation products of Clostridium daliense.

[0009] Preferably, the product includes one of the following: food and / or food additives, beverages and / or beverage additives, health products and / or health product additives, feed and / or feed additives, and drugs and / or drug additives.

[0010] The present invention also provides the application of the Clostridium daliense in the preparation of foods and / or food additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

[0011] The present invention also provides a food and / or food additive for preventing, treating or improving bodily damage caused by staying up late, wherein the food and / or food additive includes the Clostridium daliense.

[0012] The present invention also provides the application of the Clostridium daliense in the preparation of beverages and / or beverage additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

[0013] The present invention also provides a beverage and / or beverage additive for preventing, treating or improving bodily damage caused by staying up late, wherein the beverage and / or beverage additive includes the Clostridium daliense.

[0014] The present invention also provides the application of the Clostridium daliense in the preparation of yogurt for the prevention, treatment or improvement of bodily damage caused by staying up late.

[0015] The present invention also provides a yogurt for preventing, treating or improving bodily damage caused by staying up late, the yogurt comprising the Clostridium daliense.

[0016] The present invention also provides the application of the Clostridium daliense in the preparation of health products and / or health product additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

[0017] The present invention also provides a health product and / or health product additive for preventing, treating or improving bodily damage caused by staying up late, wherein the health product and / or health product additive includes the Clostridium daliense.

[0018] The present invention also provides the application of the Clostridium daliense in the preparation of probiotic agents for the prevention, treatment or improvement of bodily damage caused by staying up late.

[0019] The present invention also provides a probiotic agent for preventing, treating or improving bodily damage caused by staying up late, the probiotic agent including the Clostridium daliense.

[0020] The present invention also provides the application of the *Clostridium daliense* in the preparation of feed and / or feed additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

[0021] The present invention also provides a feed and / or feed additive for preventing, treating or improving bodily damage caused by staying up late, wherein the feed and / or feed additive includes the Clostridium daliense.

[0022] The present invention also provides the use of the Clostridium daliense in the preparation of drugs and / or drug additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

[0023] The present invention also provides a drug and / or drug additive for preventing, treating or improving bodily damage caused by staying up late, wherein the drug and / or drug additive includes the Clostridium daliense.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] This invention discloses the application of Clostridium difficile in the preparation of products that combat bodily damage caused by staying up late, which can significantly resist bodily damage caused by staying up late. The specific technical effects are as follows:

[0026] In terms of intestinal barrier protection, this strain and its products can effectively repair intestinal structural damage caused by staying up late, restore disordered colonic glands to an orderly arrangement, increase the number of goblet cells and maintain the integrity of the mucus layer, and significantly upregulate the expression of tight junction proteins such as ZO-1, Occludin, and Claudin-1, thereby enhancing the integrity and permeability regulation function of the intestinal physical barrier and effectively preventing and improving intestinal barrier damage.

[0027] In terms of anti-oxidative stress and anti-inflammation, it can activate the Nrf2 pathway, significantly increase the level of antioxidants in the body, reduce the accumulation of oxidative stress factors caused by staying up late, and enhance the body's ability to scavenge free radicals; at the same time, it can significantly downregulate the expression of inflammatory factors and inhibit systemic and brain inflammatory responses.

[0028] In terms of gut microbiota regulation, this strain and its products can improve gut microbiota imbalance caused by staying up late, regulate gut microbiota-metabolic axis homeostasis, promote the synthesis of beneficial metabolites such as short-chain fatty acids, and maintain gut microecological balance. This has important practical significance for developing functional foods, enriching the variety of fermented products, and establishing a lactic acid bacteria strain resource bank. It also provides a new method for preventing and treating bodily damage caused by staying up late.

[0029] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0030] Figure 1 For experimental group design diagram;

[0031] Figure 2 The results of each treatment group on the effects of staying up late on intestinal tissue damage;

[0032] Figure 3 The results of each treatment group on the effects of staying up late on intestinal barrier protein damage;

[0033] Figure 4 The results show the effects of each treatment group on oxidative stress damage caused by staying up late. Figure 4 In the figure, A represents the ROS statistic. Figure 4 B in the graph represents the 8-OHdG statistic. Figure 4 C in the figure represents the MDA statistical chart;

[0034] Figure 5 The results show the impact of each treatment group on the damage to the release pathway of antioxidants caused by staying up late. Figure 5 In the graph, A represents the Nrf2 statistic. Figure 5 B in the graph represents the SOD statistics. Figure 5 C in the graph represents the GSH statistics. Figure 5 D in the figure represents the HO-1 statistical chart;

[0035] Figure 6 The results show the effects of each treatment group on the level of oxidative inflammation caused by staying up late. Figure 6 In the figure, A represents the NF-κB statistical graph. Figure 6 B in the graph represents the TNF-α statistics. Figure 6 C in the graph represents the IL-1β statistics. Figure 6 D in the graph represents the IL-6 statistic. Figure 6 E in the figure represents the IL-10 statistics. Figure 6F in the figure represents the IL-17 statistics;

[0036] Figure 7 The results show the effects of different treatment groups on systemic inflammatory damage caused by staying up late. Figure 7 In the figure, A represents the CRP statistical chart. Figure 7 B in the graph represents a BDNF statistical graph;

[0037] Figure 8 The results of each treatment group on the effects of staying up late on brain inflammation and damage were presented. Figure 8 In the figure, A represents the CRP statistical chart. Figure 8 B in the figure represents the CGRP statistics chart. Figure 8 In the graph, C represents the Substance P statistic.

[0038] Figure 9 The results show the effects of each treatment group on the gut microbiota caused by staying up late. Figure 9 In the graph, A represents the Shannon index. Figure 9 B in the graph represents the Simpson index. Figure 9 In this context, C represents the number of ASVs. Figure 9 In this context, D represents the changes in the composition of the gut microbiota at the phylum level in mice from different treatment groups. Figure 9 E in the figure represents the changes in the composition of gut bacterial genera in mice under different treatment groups. Figure 9 In the figure, F represents the PCoA analysis of gut microbiota composition among different treatment groups. Figure 9 G in the figure represents the LEfSe analysis of the gut microbiota genera in mice across different treatment groups. Figure 9 H in the equation represents the LDA index in the LEfSe analysis.

[0039] Figure 10 The results of the differential analysis of serum metabolic profiles in mice in the "staying up late" group and the "yogurt" group are as follows. Figure 10 In the figure, A represents the OPLS-DA analysis of the differences in metabolic profiles between the sleep-deprivation group and the yogurt group. Figure 10 B in the diagram represents the volcano plot used to analyze differentially expressed metabolites. Figure 10 C in the table represents the TOP25 metabolites that showed significant changes. Figure 10 D in the table represents the top 15 metabolites that contributed most to distinguishing between the late-night group and the yogurt group. Figure 10 E in the diagram represents the enrichment pathway of compounds that showed significant differences between the "staying up late" group and the yogurt group. Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0042] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0043] Clostridium dalinum is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 27789 and deposit date of July 3, 2023.

[0044] Example 1

[0045] The screening and identification of Clostridium dalinum includes the following steps:

[0046] Take 10g of soil sample from Cangshan Mountain in Dali, add 225ml of sterile physiological saline, shake at 120rpm for 15min, let stand for 5min, then take 200μL of supernatant and spread it on MRS (CaCO3) medium plate, incubate at 37℃, pick colonies with clear zone formation, streak on MRS medium plate for purification, pick streak colonies for Gram staining to identify whether the strain has been successfully purified.

[0047] DNA was extracted from the bacterial strain using a Rapid Bacterial Genomic DNA Isolation Kit (Shanghai Sangon Biotech Co., Ltd.). The obtained DNA was then sent to Shanghai Sangon Biotech Co., Ltd. for DNA sequencing to obtain strain information.

[0048] Example 2

[0049] The *Clostridium daliense* obtained in Example 1 was inoculated into MRS liquid medium and cultured at 37°C with shaking at 120 rpm for 24 h. The culture was then removed, poured into sterile 50 mL centrifuge tubes, and centrifuged at 8000 rpm for 10 min, discarding the supernatant. Sterile physiological saline was added for washing, followed by centrifugation at 8000 rpm for 10 min, discarding the supernatant, and repeating the washing process three times. An appropriate volume of physiological saline was added to adjust the OD value of the bacterial culture to 0.60 ± 0.02, thus obtaining the *Clostridium daliense* bacterial culture.

[0050] According to the "General Experimental Tutorial on Microbiology", take 1000mL of pure milk, add 6% (w / v) white sugar, add 1% of Clostridium difficile live bacterial solution, place it in an environment of 42℃ and ferment for 4h, then put it in a refrigerator at 4℃ for 48h to obtain fermented yogurt for later use.

[0051] The effectiveness of the yogurt obtained in Example 2 was verified through the following experiments:

[0052] The following experiments were conducted using Figure 1 The experimental scheme shown is used to group the samples.

[0053] 1. Histological analysis of mice:

[0054] Mice were euthanized by cervical dislocation, and the abdomen was opened in a "V" shape to expose the abdominal cavity. The colon was then harvested, and the samples were processed as follows: Three mice were randomly selected from each group, and an intestinal segment was harvested 1 cm below the cecum. The segments were fixed in 4% paraformaldehyde and stored at room temperature for tissue section preparation. Hematoxylin-Eosin (HE) staining was used to observe the tissue sections, and the results are as follows. Figure 2 As shown.

[0055] Depend on Figure 2 The study showed that the mice in the "staying up late" group had disordered colonic gland arrangement, reduced goblet cells, and submucosal fibrosis. In contrast, the mice in the "yogurt" group had nearly normal colonic gland arrangement, dense goblet cells, and an intact mucus layer, almost completely restoring gland morphology and submucosal thickness, suggesting that yogurt can effectively prevent intestinal structural damage caused by staying up late.

[0056] 2. Immunohistochemical analysis in mice:

[0057] Paraffin sections of mouse colon tissue were dewaxed, dehydrated with anhydrous ethanol, and then hydrated with distilled water. Before antigen binding, the sections were microwaved to boiling in citrate buffer (pH 6.0), cooled, and washed with PBS. Then, endogenous peroxidase was blocked by drawing circles with an immunohistochemical pen and adding 3% hydrogen peroxide, incubated at room temperature in the dark for 25 min, and then washed with PBS. Next, serum blocking was performed using 10% rabbit serum or 3% BSA for 30 min. After the first round of incubation with primary antibody (4°C overnight), HRP-labeled secondary antibody was added and incubated for 50 min, with the signal amplified by iF555-TSA, and incubated in the dark for 10 min. After removing the antibody complex using microwave treatment, the above steps were repeated for the second and third rounds of labeling. The second round used iF488-TSA to amplify the signal, and the third round used iF647-labeled fluorescent secondary antibody. Finally, the sections were stained with DAPI (10 min), quenched for 5 min of autofluorescence, rinsed with running water, and mounted. Panoramic imaging of fluorescence signals was performed using a digital scanning system to analyze the expression intensity and distribution of positive cells or regions. The results are as follows: Figure 3 As shown.

[0058] Depend on Figure 3The fluorescence intensity of the tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon of mice in the sleep-deprivation group was significantly reduced, indicating impaired physical integrity and permeability regulation of the intestinal barrier. The expression of ZO-1, Occludin, and Claudin-1 in the yogurt group was close to that in the blank control group, suggesting that its components may reduce the direct damage to tight junction structures caused by sleep deprivation by enhancing intercellular connection stability in advance.

[0059] 3. Analysis of oxidative stress factors, oxidative inflammatory factors, and biochemical indicators related to inflammatory damage in mouse serum:

[0060] Oxidative stress factors, oxidative inflammatory factors, and inflammatory damage were detected using commercially available kits. The indicators detected included: serum ROS, MDA, and 8-OHdG to assess the level of oxidative stress; serum NF-κB, TNF-α, IL-1β, IL-6, IL-10, and IL-17 to measure the level of oxidative inflammatory factors; serum Nrf2, HO-1, SOD, and GSH to assess the body's antioxidant capacity; serum CRP and BDNF to evaluate the degree of inflammatory damage; and brain gray matter CRP, CGRP, and Substance P to evaluate the degree of brain inflammatory damage. All indicators were analyzed according to the enzyme-linked immunosorbent assay (ELISA) kit instructions. Optical density (OD) values ​​were measured using a microplate reader (450 nm), and the concentrations of each indicator were calculated based on a standard curve. Results are as follows: Figures 4-8 As shown.

[0061] Depend on Figure 4 The results showed that the serum ROS, 8-OHdG, and MDA levels in the sleep-deprivation group were increased by 1.08% (P<0.001), 101.98% (P<0.001), and 78.67% (P<0.001), respectively, compared to the control group, indicating that staying up late can induce oxidative stress in the body. Meanwhile, mice fed yogurt simultaneously showed a 4.99% (P<0.001) decrease in serum ROS levels, a 43.32% (P<0.001) decrease in 8-OHdG levels, and an 11.85% (P<0.001) decrease in MDA levels compared to the sleep-deprivation group. These experimental results indicate that yogurt prepared by Clostridium difficile fermentation exhibits good antioxidant capacity and can effectively alleviate oxidative stress damage caused by staying up late.

[0062] Depend on Figure 5The results showed that the activities of Nrf2, SOD, GSH, and HO-1 in the sleep-deprivation group were reduced by 25.30% (P<0.001), 32.47% (P<0.001), 35.74% (P<0.001), and 49.17% (P<0.001), respectively, compared with the blank control group. This indicates that sleep-deprivation exposure inhibited or weakened the expression of antioxidant-related factors and enhanced oxidative stress response. Meanwhile, mice fed yogurt simultaneously showed, compared with the sleep-deprivation group, a 41.24% (P<0.001) increase in serum Nrf2 level, a 68.11% (P<0.001) increase in SOD level, a 33.45% (P<0.001) increase in GSH level, and a 46.35% (P<0.001) increase in HO-1 level. Experimental results show that yogurt prepared by Clostridium difficile fermentation can upregulate the levels of Nrf2, SOD, GSH, and HO-1 in the body, thereby reducing oxidative stress and minimizing damage to the body, and has a good antioxidant effect.

[0063] Depend on Figure 6 The results showed that the levels of NF-κB, TNF-α, IL-1β, IL-10, IL-6, and IL-17 in the sleep-deprivation group increased by 164.05% (P<0.001), 194.99% (P<0.001), 737.06% (P<0.001), 231.61% (P<0.001), 16.77% (P<0.001), and 557.45% (P<0.001), respectively, compared with the blank control group. This indicates that staying up late induces or aggravates the inflammatory response. Mice fed yogurt simultaneously showed significantly lower levels of NF-κB (47.52%, P<0.001), TNF-α (16.09%, P<0.001), IL-1β (40.33%, P<0.001), IL-10 (20.02%, P<0.001), IL-6 (10.61%, P<0.001), and IL-17 (38.28%, P<0.001) compared to the group that stayed up all night. These results indicate that yogurt fermented with *Clostridium difficile* can reduce oxidative inflammation caused by staying up all night by downregulating the level of inflammatory responses, demonstrating a strong anti-inflammatory effect.

[0064] Depend on Figure 7The results showed that the serum CRP and BDNF levels in the "staying up late" group were increased by 127.39% (P<0.001) and 1036.9% (P<0.001), respectively, compared to the control group. These changes indicated that the mice had developed inflammatory damage. Meanwhile, mice fed yogurt showed a 45.98% (P<0.001) decrease in serum CRP levels and a 31.65% (P<0.001) decrease in BDNF levels compared to the "staying up late" group. These experimental results demonstrate that yogurt prepared using *Clostridium difficile* fermentation can effectively reduce inflammatory damage caused by staying up late, highlighting its potential value in combating inflammatory symptoms.

[0065] Depend on Figure 8 The results showed that the levels of CGRP, CRP, and Substance P in the brains of mice in the "staying up late" group were increased by 576.34% (P<0.001), 111.99%, and 193.27% (P<0.001), respectively, compared to the control group. These changes indicate that the mice's brains had already suffered inflammatory damage. Meanwhile, mice fed yogurt showed a 49.09% (P<0.001) decrease in serum CGRP levels, a 42.51% (P<0.001) decrease in CRP levels, and a 28.81% (P<0.001) decrease in BDNF levels compared to the "staying up late" group. These experimental results indicate that yogurt prepared using *Clostridium difficile* fermentation can effectively reduce brain damage caused by staying up late, especially inflammatory damage, highlighting its potential in combating brain damage caused by staying up late.

[0066] 4. Analysis of mouse gut microbiota:

[0067] Multidimensional microbiome analysis was used to investigate the effects of sleep deprivation on the gut microbiota of mice and its regulatory role in yogurt fermented with Clostridium daliense. The results are as follows: Figure 9 As shown.

[0068] Depend on Figure 9 It can be seen that staying up late did not significantly alter Shannon's (P = 0.98) Figure 9 In the A) Simpson index (P>0.99) Figure 9 (B in the text), but significantly altered the observed ASV number (P<0.001) ( Figure 9 The C in the figure indicates that staying up late did not significantly disrupt the uniformity of the gut microbiota, but it significantly altered the diversity in the gut.

[0069] At the phylum level, Bacteroidota (37.27%), Bacillota (31.69%), Verrucomicrobiota (1.18%), Campylobacterota (12.28%), Thermodesulfobacteriota (6.11%), and Deferribacterota (5.35%) constituted the dominant flora in the sleep-deprivation group. The content of Bacteroidota decreased by 48.09% (P<0.001) and the content of Bacillota decreased by 49.08% (P<0.001) compared to the control group. These two phyla are common bacteria in the gut microbiota and are closely related to the integrity of the intestinal barrier and immune function; their reduction may exacerbate intestinal barrier damage induced by sleep deprivation. In the yogurt treatment group, the contents of Bacteroidota and Bacillota increased by 98.53% and 16.65% respectively compared to the sleep-deprivation group. Figure 9 (D in the middle).

[0070] At the species level, the predominant species in the "staying up late" group were Akkermansia muciniphila (1.17%), Helicobacterrodentium (10.97%), Mucispirillum schaedleri (6.14%), Lactobacillus vaginalis (0.85%), and Bacteroides sartorii (0.20%). Among these, the contents of Akkermansia muciniphila and Bacteroides sartorii decreased by 141.04% and 28.63% respectively compared to the control group (P<0.001). These two are common probiotics in the gut, crucial for gut microbiota regulation and maintaining bodily health; their reduction may exacerbate gut microbiota imbalance and metabolic disorders, leading to damage to health. In the yogurt treatment group, the contents of Akkermansia muciniphila and Bacteroides sartorii increased by 1858.05% and 175.20% respectively compared to the "staying up late" group. Figure 9 (E in the text)

[0071] β-diversity analysis showed significant differences in gut microbiota among different treatment groups (R0). 2 =0.4767, P<0.001)( Figure 9 (F in the text); LEfSe analyzed a total of 320 specific genera (F in the text); Figure 9 G and Figure 10Among the H results, 141 mice were found to have been kept up late, indicating that staying up late caused significant changes in the composition of the gut microbiota in mice. 53 mice were fed yogurt, which also demonstrates that yogurt feeding has a significant impact on the composition of the gut microbiota.

[0072] In summary, yogurt fermented with Clostridium daliense can counteract the damage to the body caused by staying up late by altering the structure of the intestinal flora and affecting the content of beneficial and harmful bacteria.

[0073] 5. Analysis of mouse serum metabolic profiles:

[0074] To further explore the intervention mechanism of yogurt prepared from *Clostridium difficile* on the damage caused by staying up late in mice, this study used non-targeted metabolomics technology to analyze changes in serum metabolic phenotypes in mice. The results are as follows: Figure 10 As shown.

[0075] Depend on ​ As shown, yogurt prepared from *Clostridium difficile* significantly modulates the serum metabolic profile phenotype of mice. Compared to the sleep-deprivation group, 2448 significantly different compounds were identified in the yogurt group, of which 739 were upregulated and 1710 were downregulated. Specifically, L-Lactic acid, Ethylbenzoate, Uric acid, and Resorcinol showed significant changes. L-Lactic acid was significantly increased in the yogurt-fed group (P<0.001), confirming that active bacteria such as lactic acid bacteria in the mouse gut were significantly enriched after yogurt feeding. The short-chain fatty acids produced by these bacteria can maintain metabolic and immune balance and inhibit pathogen proliferation. The significant decreases in Ethylbenzoate and Resorcinol (P<0.001) indicate that yogurt feeding inhibits toxic substances in the gut microbiota, possibly by inhibiting the enrichment of toxin-producing bacteria in the gut. In addition, it also enhances the expression of intestinal tight junction proteins, reducing the possibility of leaky gut.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of Clostridium dalinum in the preparation of products for the prevention, treatment or improvement of bodily damage caused by staying up late.

2. The application according to claim 1, characterized in that, The products include one of the following: food and / or food additives, beverages and / or beverage additives, health products and / or health product additives, feed and / or feed additives, and drugs and / or drug additives.

3. The use of Clostridium daliense as described in claim 1 in the preparation of foods and / or food additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

4. A food and / or food additive for preventing, treating, or improving bodily damage caused by staying up late, characterized in that, The food and / or food additives include Clostridium daliense as described in claim 1.

5. The use of Clostridium daliense as described in claim 1 in the preparation of beverages and / or beverage additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

6. A beverage and / or beverage additive for preventing, treating, or improving bodily damage caused by staying up late, characterized in that, The beverage and / or beverage additives include Clostridium daliense as described in claim 1.

7. The use of Clostridium daliense as described in claim 1 in the preparation of health products and / or health product additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

8. A health product and / or health product additive for preventing, treating, or improving bodily damage caused by staying up late, characterized in that, The health products and / or health product additives include the Clostridium daliense as described in claim 1.

9. The use of Clostridium daliense as described in claim 1 in the preparation of feed and / or feed additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

10. A feed and / or feed additive for preventing, treating, or improving bodily damage caused by staying up late, characterized in that, The feed and / or feed additives include Clostridium daliense as described in claim 1.

11. The use of Clostridium difficile as described in claim 1 in the preparation of medicaments and / or pharmaceutical additives for the prevention, treatment or improvement of bodily damage caused by staying up late.

12. A drug and / or drug additive for preventing, treating, or improving bodily damage caused by staying up late, characterized in that, The drug and / or drug additives include Clostridium daliense as described in claim 1.

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