Bacillus subtilis strain JQ-14 and application thereof

Abstract

The present application relates to the technical field of microorganism, in particular to a bacillus subtilis JQ-14 and application thereof, and provides the bacillus subtilis JQ-14, which is preserved in the China General Microbiological Culture Collection Center on May 22, 2026, and the preservation number is CGMCC No. 38830, and the preservation address is No. 3, Xibahe West Road, Chaoyang District, Beijing City.The ADH activation rate of the strain is up to 24%, the degradation rate of 10% volume fraction of ethanol is 42%, the alcohol in the body can be quickly metabolized, and the inebriety discomfort can be relieved;the mouse ALT and AST levels can be significantly reduced, the MDA can be down-regulated, the enzyme activity of SOD and GSH-Px can be improved, the alcohol-induced liver tissue peroxidation damage can be improved, and the intestinal flora regulation capacity is significant, and the strain can survive in the environment of 30-37 DEG C and pH 2-7.5, and the process controllability is stronger.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus subtilis JQ-14 and its applications. Background Technology

[0002] Alcohol, an indispensable beverage at the dinner table, has seen its consumption increase year by year. The liver, a vital organ for alcohol metabolism, is directly damaged by ethanol and acetaldehyde over extended periods, leading to alcoholic liver disease, fatty liver, or liver fibrosis. In severe cases, it can cause cirrhosis, ascites, and even liver cancer. Furthermore, prolonged and excessive alcohol consumption not only disrupts the tight junction proteins between intestinal flora and intestinal epithelial cells, causing immune deficiency and impaired digestion and absorption, but also induces the accumulation of acetaldehyde and other toxic substances in the liver. This causes endoplasmic reticulum stress and cell apoptosis, reducing antioxidant and detoxification capabilities, ultimately leading to liver disease.

[0003] Currently, there are various hangover remedies available, but they cannot directly break down alcohol in the gastrointestinal tract. Instead, they work by reducing alcohol absorption in the gastrointestinal tract or by relying on the body's own endogenous enzyme system to assist in alcohol metabolism. However, the regulation of the activity of enzymes involved in the various stages of ethanol metabolism in the human body is extremely complex. It is difficult to simply rely on exogenous drugs to improve enzyme levels and activity, resulting in relatively weak effects and difficulty in achieving a rapid hangover cure. Summary of the Invention

[0004] In view of this, the present invention provides a strain of Bacillus subtilis JQ-14 and its applications. This strain can efficiently activate alcohol dehydrogenase, accelerate the decomposition of ethanol in the body, and at the same time, it can protect the liver, repair liver damage, and regulate the diversity of intestinal microbiota.

[0005] To solve the above technical problems, the present invention provides a strain of Bacillus subtilis (B. subtilis). Bacillus subtilis JQ-14, this strain was deposited on May 22, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38830, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The strain JQ-14 provided by this invention exhibits an activation rate of up to 24% for ADH and an in vitro degradation rate of up to 42% for 10% volume fraction ethanol, significantly improving the degradation rate of ethanol in vivo, shortening the duration of intoxication, and reducing alcohol accumulation in the body. Moreover, compared with existing Bacillus subtilis strains, this strain can significantly reduce ALT and AST levels in mice, inhibit hepatocyte damage and leakage, and has a good liver-protective effect. It can also significantly downregulate lipid peroxide MDA, increase the activity of SOD and GSH-Px antioxidant enzymes, improve alcohol-induced liver tissue peroxidation damage, improve liver oxidative stress levels, and resist alcohol-induced liver oxidative stress. In addition, this strain can optimize intestinal flora imbalance after alcohol damage, increase the abundance of beneficial bacteria, inhibit the colonization of pro-inflammatory microorganisms, and has a significant ability to regulate the intestinal microbiota, without the toxic side effects of chemical drugs, making it highly safe for consumption.

[0007] The strain JQ-14 provided by this invention can adapt to a growth environment of 30~37℃ and pH 2~7.5, has stronger process controllability, and the product activity stability is better than that of existing common strains.

[0008] A second aspect of the present invention provides a microbial inoculant comprising the aforementioned Bacillus subtilis JQ-14.

[0009] A third aspect of the present invention provides a hangover remedy product comprising the aforementioned Bacillus subtilis JQ-14 or a microbial agent.

[0010] The hangover relief products provided by this invention include, but are not limited to, food, health products or medicines. The product form can be a liquid oral preparation or a solid oral preparation, including but not limited to solutions, suspensions, ordinary tablets or capsules.

[0011] A fourth aspect of the present invention provides a liver-protecting product comprising the aforementioned Bacillus subtilis JQ-14 or a microbial agent.

[0012] The liver protection products provided by this invention include, but are not limited to, food, health products or medicines. The product form can be a liquid oral preparation or a solid oral preparation, including but not limited to solutions, suspensions, ordinary tablets or capsules.

[0013] The fifth aspect of the present invention provides a product for regulating intestinal microbiota, comprising the above-mentioned Bacillus subtilis JQ-14 or microbial agents.

[0014] The products for regulating intestinal microbiota provided by this invention include, but are not limited to, food, health products or pharmaceuticals. The product form can be a liquid oral preparation or a solid oral preparation, including but not limited to solutions, suspensions, ordinary tablets or capsules.

[0015] The strain JQ-14 provided by this invention can not only efficiently degrade ethanol, but animal experiments have also confirmed that it can effectively alleviate alcohol-induced intestinal barrier dysfunction, liver dysfunction, oxidative stress and intestinal flora imbalance.

[0016] The sixth aspect of the present invention provides the application of the above-mentioned Bacillus subtilis JQ-14 in the preparation of products that enhance ADH activity and reduce alcohol concentration.

[0017] Optionally, the product that enhances ADH activity and reduces alcohol concentration is a hangover remedy.

[0018] The seventh aspect of the present invention provides the use of the above-mentioned Bacillus subtilis JQ-14 in the preparation of products that reduce ALT, AST and MDA and increase SOD and GSH-Px enzyme activities.

[0019] Optionally, the product that reduces ALT, AST, and MDA and increases SOD and GSH-Px enzyme activity is a liver-protecting product.

[0020] The eighth aspect of the present invention provides the application of the above-mentioned Bacillus subtilis JQ-14 in the preparation of products that regulate intestinal microbiota. Attached Figure Description

[0021] Figure 1 The images show colony and cell images of Bacillus subtilis JQ-14 provided by this invention, wherein (a) is a colony morphology image and (b) is a cell morphology image after Gram staining.

[0022] Figure 2 The phylogenetic tree of Bacillus subtilis JQ-14 provided by the present invention.

[0023] Figure 3 These are photographs of liver tissues from the CON group, ETH group, and JQ-14 group after HE staining, observed under an optical microscope in Example 3 of the present invention.

[0024] Figure 4 These are photographs of colon tissues from the CON group, ETH group, and JQ-14 group after HE staining, observed under an optical microscope in Example 3 of this invention.

[0025] Figure 5 This is a bar chart showing the gut microbiota α diversity index of the CON group, ETH group, and JQ-14 group in Example 3 of the present invention.

[0026] Figure 6 This is a stacked bar chart of the phylum-level species composition in Embodiment 3 of the present invention.

[0027] Figure 7 This is a stacked bar chart of the horizontal species composition in Embodiment 3 of the present invention.

[0028] Figure 8 This is a stacked bar chart of the species composition at the level of the present invention in Embodiment 3. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0030] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art and should not be interpreted in an idealized or overly formal sense unless specifically defined.

[0031] The essence of sobering up involves using alcohol dehydrogenase (ADH) to oxidize ethanol into acetaldehyde, which is then oxidized into acetic acid by acetaldehyde dehydrogenase. Acetic acid enters the bloodstream and is metabolized into carbon dioxide, water, and energy. Therefore, the main function of sobering up is to activate the production of ADH in the body, increase ADH activity, accelerate the oxidative decomposition of ethanol in the body, reduce the concentration of ethanol in the body, shorten the duration of intoxication, and alleviate the discomfort caused by intoxication. Although experimental studies have confirmed that Bacillus subtilis has functions such as regulating the intestinal microbiota, enhancing the host's intestinal mucosal immune function, promoting the host's liver enzyme activity, and improving alcohol-induced liver oxidative damage. However, the activation rate of endogenous ADH in the fermentation products of common wild Bacillus subtilis is only 7%~12%, which cannot efficiently enhance the activity of ADH in the body. The effect of accelerating the metabolism of ingested ethanol is limited, and the onset of alcohol detoxification is slow. In animal models of alcoholic liver disease, the existing Bacillus subtilis can only slightly downregulate alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA) in the liver, and the upregulation of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) is limited. It is difficult to significantly reverse the oxidative necrosis of hepatocytes caused by alcohol, and the liver protection effect is weak and the liver protection and antioxidant effect is insufficient. Moreover, the suitable growth range of conventional Bacillus subtilis is relatively narrow. When deviating from the suitable temperature and pH, there are problems such as spore formation and a significant decrease in the secretion of active metabolites. The batch stability of the finished product is poor and it is constrained by the culture conditions.

[0032] In view of this, the present invention provides a Bacillus subtilis strain JQ-14 and its applications. This strain can achieve a degradation rate of 42% for 10% volume fraction ethanol and an ADH activation rate as high as 24%, enabling it to rapidly metabolize alcohol in the body. At the same time, this strain can significantly reduce ALT and AST levels, inhibit hepatocyte damage and leakage, significantly downregulate lipid peroxide MDA, enhance the activity of SOD and GSH-Px antioxidant enzymes, improve alcohol-induced liver tissue peroxidation damage, and has a wider suitable growth range, adapting to a growth environment of 30~37℃ and pH 2~7.5. The process controllability is stronger, and the activity stability of the strain is significantly better than other strains.

[0033] The culture media used in this invention include the following: NA medium: 2 g beef extract, 10 g peptone, 10 g sodium chloride, 15-20 g agar (2%), bring to a final volume of 1 L distilled water, pH 7.0.

[0034] Fermentation medium: 2 g beef extract, 10 g peptone, 10 g sodium chloride, and diluted to 1 L of distilled water.

[0035] Initial screening medium: 0.1 g sodium chloride, 0.3 g magnesium sulfate, 5 g ammonium sulfate, 1.0 g potassium dihydrogen phosphate, 0.01 g ferric chloride, 22 g agar, diluted to 1 L with distilled water, pH 7.0. After sterilization, cool the medium to 50°C, add 8% anhydrous ethanol, and set aside.

[0036] All the above culture media were sterilized in a high-pressure steam sterilizer at a temperature of 121°C for 20 minutes.

[0037] Unless otherwise specified, the chemical reagents and equipment used in the following embodiments of the present invention are all commercially available products.

[0038] Example 1 This embodiment provides the isolation, screening, and identification of Bacillus subtilis JQ-14, including: (1) Three sample materials were obtained from traditional fermented foods (koji) and the brewing environment. 20 g of each sample was placed in 190 mL of sterile water and cultured on a shaker at 37℃ and 180 rpm / min for 2-3 h. The cultured samples were then serially diluted 10-fold with sterile water. -6 The serially diluted culture medium was spread onto NA medium plates and incubated at 37°C for 24 h. Single colonies were picked, streaked onto plates, and stored on slant plates for later use.

[0039] (2) Initial screening: Microbial isolation and purification were carried out on three different samples. After serial dilution, plate plating, single colony picking and repeated purification, 45 pure culture strains were obtained from each sample, for a total of 135 pure strains. Using ethanol tolerance as the screening index, the 135 purified strains were inoculated into an initial screening medium containing 8% ethanol and cultured in a constant temperature incubator at 37℃. Based on the comprehensive judgment of colony growth and tolerance performance, 30 ethanol-tolerant dominant strains were finally screened and entered the secondary screening stage to determine their ethanol degradation rate.

[0040] Secondary screening: The ethanol-tolerant strains obtained from the initial screening were inoculated into fermentation medium (pH = 7) and cultured at 37℃ and 180 rpm / min on a shaker for 12 h to obtain seed culture. The seed culture was inoculated at a rate of 5% (6 × 10⁻⁶). 8 The bacterial culture (CFU / mL) was inoculated into a 10% ethanol fermentation medium (pH = 7) and cultured at 37 ℃ for 24 h, with a 10% ethanol fermentation broth as a blank control. 1 mL of the bacterial culture was centrifuged at 10000 rpm for 10 min, and 100 μL of the supernatant was collected to determine the ethanol degradation rate (%) using the potassium dichromate method. The formula for calculating the ethanol degradation rate is as follows: , in, C 0 represents the volume fraction (%) of ethanol in the blank control. C The volume fraction (%) of ethanol in the experimental group.

[0041] Among the 30 ethanol-resistant dominant strains, strain 14 had the most outstanding alcohol degradation ability, with an ethanol degradation rate of up to 42%, and was named JQ-14.

[0042] (3) The strain JQ-14 with the best ethanol degradation effect was identified: Morphological observation and physiological and biochemical identification: Biochemical identification of the isolated pure single colonies was performed according to the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification".

[0043] Systematic morphological observations were conducted on the selected target strain JQ-14, such as... Figure 1 As shown in (a), this strain forms white colonies on solid culture medium, with slightly irregular edges and a slightly sticky texture; after Gram staining and microscopic examination under an oil immersion microscope, as shown... Figure 1 As shown in (b), the bacteria have a typical purple rod-shaped morphology. Based on the staining results, strain JQ-14 can be identified as a Gram-positive bacillus.

[0044] The results of the physiological and biochemical tests are shown in Table 1. The results of the physiological and biochemical indicators were compared with those in the *Manual of Systematic Identification of Common Bacteria* and the *Bergey's Manual of Bacterial Identification*, and combined with morphological identification, the results showed that strain JQ-14 conformed to the characteristics of Bacillus subtilis. Therefore, strain JQ-14 was preliminarily identified as Bacillus subtilis.

[0045] Table 1 Physiological and biochemical indicators of strain JQ-14 Molecular biological identification: DNA was extracted from the bacterial culture according to the instructions in the "Bacterial Genomic DNA Rapid Extraction Kit," and the obtained DNA was amplified by PCR. The PCR-amplified genome was sent to the company for sequencing. The sequenced bacterial DNA sequence was BLASTed on NCBI, and a phylogenetic tree was constructed using Mega 6.0 software, such as... Figure 2 As shown.

[0046] The 16S rRNA gene sequence of JQ-14 (as shown in SEQ No: 1) is extremely similar to the 16S rRNA sequence of Bacillus subtilis recorded in the BLAST database, with a similarity exceeding 99%. After comprehensively considering the morphological characteristics, physiological and biochemical identification results, and 16S rRNA sequencing and phylogenetic analysis results, strain JQ-14 was identified as Bacillus subtilis.

[0047] Strain JQ-14 has been 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. 38830 and deposit date of May 22, 2026.

[0048] As shown in Table 1, strain JQ-14 can form spore structure characteristics. The thick-walled structure of the spores has excellent stress resistance and can resist the corrosion of strong gastric acid. After oral administration, it can successfully reach the intestine and colonize to exert its effects, and can exert long-term metabolic and regulatory effects. In contrast, probiotics without spore structure are easily inactivated by gastric acid, affecting intestinal utilization.

[0049] (4) The degradation rate of JQ-14 under different pH conditions was investigated. The isolated and purified JQ-14 was inoculated into fermentation medium (pH = 7) and cultured at 37℃ and 180 rpm / min for 12 h as seed culture. The seed culture was inoculated at a rate of 5% (6 × 10⁻⁶). 8CFU / mL of the bacterial culture was inoculated into fermentation media with different pH values ​​(pH = 3, 4, 5, 6, 7) containing 10% ethanol and cultured at 37 ℃ for 24 h, with 10% ethanol broth as a blank control. Then, 1 mL of the bacterial culture was centrifuged at 10000 rpm for 10 min, and 100 μL of the supernatant was collected to determine the ethanol degradation rate (%) using the potassium dichromate method. The ethanol degradation rate was measured under different pH conditions. The formula for calculating the ethanol degradation rate is as follows: Where C0 is the volume fraction (%) of ethanol in the blank control; C is the volume fraction (%) of ethanol in the experimental group.

[0050] The alcohol degradation rate is 21% at pH = 3, 26% at pH = 4, 30% at pH = 5, 37% at pH = 6, and 42% at pH = 7.

[0051] (5) Investigate the survival ability of strain JQ-14 under different pH conditions. Accurately weigh 0.1 g of the lyophilized bacterial strain powder, add 10 mL of pre-cooled sterile PBS (pH 7.0), and shake thoroughly to prepare a bacterial suspension. Take 1 mL of the bacterial suspension and add 9 mL of sterile PBS buffer at pH 2.0, pH 3.0, pH 4.0, and pH 5.0, respectively, with pH 7.0 PBS as the control group. The suspension was subjected to shaking stress at 37℃ and 120 rpm. Samples were taken at 0 h, 1 h, 2 h, and 3 h, serially diluted, and plated for viable bacterial count. Each group had three biological replicates, and the average value was calculated. The results are shown in Table 2.

[0052] Table 2 As shown in Table 2, the survival rate of the strain decreased with decreasing pH and prolonged stress time. The strain maintained a high survival rate at pH 5.0; the survival rate decreased at pH 4.0; the survival rate was greater than 60% after 2 hours at pH 3.0; and the survival rate was greater than 40% after 3 hours at pH 2.0, indicating that the strain has a certain tolerance to strongly acidic environments.

[0053] Example 2 This embodiment provides the application of strain JQ-14 in enhancing alcohol dehydrogenase (ADH) activity.

[0054] JQ-14 standard slant culture was inoculated onto NA solid plates and incubated at 37 ℃ for 24 h to obtain single colonies. Typical single colonies were picked and transferred to NB liquid medium and cultured on a shaker at 37 ℃ and 200 r / min for 4 h until the OD600 of the culture reached 0.5, which was used as the primary seed culture. The primary seed culture was inoculated into fresh NB liquid medium at an inoculation rate of 2% (v / v) and cultured on a shaker under the same conditions (37 ℃, 200 r / min) for 24 h to obtain the fermentation culture. The fermentation broth was centrifuged at 4 ℃ and 8000 r / min for 10 min, the supernatant was discarded, and the bacterial precipitate was collected. The bacterial precipitate was resuspended in sterile physiological saline, and centrifuged and washed twice more under the same conditions to remove residual culture medium. The washed bacterial cells were placed in a vacuum freeze dryer and freeze-dried for 48 h. The cells were then ground and pulverized to obtain JQ-14 bacterial powder, which was sealed and stored at low temperature for later use. Before use, the bacterial suspension was reconstituted with sterile physiological saline to prepare a bacterial suspension of the corresponding concentration.

[0055] Accurately weigh 0.1 g of JQ-14 lyophilized bacterial powder, add 10 mL of pre-cooled PBS buffer (pH 7.0) to prepare a bacterial powder suspension, shake in an ice-water bath for 60 min, centrifuge at 4 ℃ and 8000 r / min for 10 min, collect the supernatant and filter it through a 0.22 μm aqueous filter membrane to obtain the test sample solution, and keep it on ice for later use; add 1.5 mL of sodium pyrophosphate buffer (pH 8.5), 1.0 mL of NAD⁺ solution, 0.5 mL of anhydrous ethanol, and 0.1 mL of the test sample solution to a centrifuge tube in sequence, mix well, and incubate at 37 ℃ for 5 min; set up a sterile control group (an equal volume of PBS buffer replaced the test sample solution), a JQ-14 test group, and a substrate blank control group (without anhydrous ethanol, this control group used distilled water instead of alcohol dehydrogenase solution to eliminate sample light absorption interference), and set up 3 biological replicates for each group. After preheating, 0.1 mL of alcohol dehydrogenase solution was added to both the sterile control group and the JQ-14 experimental group to initiate the reaction. Absorbance was recorded every 30 seconds at 340 nm wavelength under 37℃ conditions for 5 minutes. The zero point was set using a control tube. Based on the data, the linear change range in the initial stage of the reaction was selected, and the absorbance change per minute ΔA340 / min was calculated. Enzyme activity calculation formula: , NADH molar extinction coefficient ε = 6.22 mM⁻¹·cm⁻¹; cuvette path length d = 1 cm; V 总 V represents the total volume of the enzyme-catalyzed reaction system. 酶 This indicates the volume of ADH enzyme solution added.

[0056] Activation rate calculation formula: , Calculations show that the JQ-14 bacterial extract has an in vitro activation rate of up to 24.0% for alcohol dehydrogenase.

[0057] Example 3 This embodiment provides the application of strain JQ-14 in reducing liver damage, alleviating liver dysfunction and oxidative stress, and regulating the gut microbiota. Mice were selected for animal experiments.

[0058] Experimental animals: SPF-grade male ICR mice, 6 weeks old, weighing approximately 25 g, purchased from a reputable laboratory animal center; Housing environment: 12 h light / 12 h dark cycle, ambient temperature 25℃, relative humidity 56%±2%.

[0059] Adaptive feeding: Mice were given free access to regular feed and sterile water for 5 days of pre-feeding acclimatization. After excluding individuals with abnormal conditions, they were divided into three groups of 12 mice each: blank control group (CON), alcohol model group (ETH), and JQ-14 strain treatment intervention group (JQ-14).

[0060] An alcoholic liver injury model was established: The blank control group was administered 12 mL / kg of normal saline daily by gavage; the other two groups, except for the blank group, were administered 12 mL / kg of 56% vol baijiu (Chinese liquor) daily by gavage to establish an acute alcohol injury model. Four hours after alcohol gavage, mice in the JQ-14 treatment group were administered 1×10... 9 JQ-14 bacterial suspension (10 mL / kg) at cfu / mL was administered for 12 consecutive days. Mice were weighed every 3 days during the experiment, and the gavage volume was adjusted accordingly. The survival status of the mice was observed daily, and the number of deaths in each group was recorded. At the end of the experiment, the mortality rates of each group were calculated (blank control group: 0%; alcohol model group: 52.94%; treatment intervention group: 25%). It was observed that the mortality rate of mice treated with JQ-14 was 27.94 percentage points lower than that of the alcohol model group. At the end of the experiment, fresh fecal contents were aseptically collected from the mice, aliquoted, and frozen at -80 ℃ for subsequent high-throughput sequencing analysis of intestinal microorganisms' 16S rRNA.

[0061] After the last administration, mice were fasted for 12 hours but allowed free access to water. Before collection, mice were weighed, and blood was collected from the eyeballs in 1.5 mL centrifuge tubes. After standing at room temperature for 1 hour, the blood was centrifuged at 3500 r / min for 10 min in a 4 ℃ refrigerated high-speed centrifuge. The supernatant was collected by pipette and used as mouse serum for further analysis. After blood collection from the eyeballs, the mice were fixed in an operating table, their fur was trimmed, and the area was disinfected. The skin on both sides of the mouse's abdomen was lifted with hemostatic forceps, and the abdominal skin was carefully incised with a scalpel to expose the intact liver. The liver was quickly removed to observe morphological changes, rinsed twice with PBS, blotted dry with filter paper, and then weighed to calculate the liver index.

[0062] The liver index results for each group are shown in Table 3.

[0063] Liver index (%) = Liver weight (g) / Mouse weight (g) × 100%.

[0064] Table 3 As shown in Table 3, compared with the blank control group, the mice in the alcohol model group had a significantly lower final weight and a significantly higher liver index; while after feeding with Bacillus subtilis JQ-14, the mice's final weight was effectively restored and the liver index was significantly reduced, indicating that Bacillus subtilis JQ-14 can significantly improve the growth inhibition and liver enlargement in mice caused by alcohol intake.

[0065] After dissecting the mice, a 1 cm × 1 cm section of liver tissue was taken from the middle of the left lobe of the liver. Simultaneously, the distal colon tissue was dissected, and a 1 cm segment of colon was harvested. Both tissue samples were fixed in 10 times their volume of 4% paraformaldehyde, and after routine dehydration, paraffin embedding, and serial sectioning, hematoxylin-eosin (HE) staining was performed. The sections were mounted with neutral resin, and the degree of pathological damage to the liver in each group was observed under a light microscope (e.g., ...). Figure 3 (as shown) and changes in the structure of the colonic mucosal barrier tissue (such as...) Figure 4 (As shown). The remaining liver tissue was cut into small pieces, frozen in liquid nitrogen, and then transferred to an ultra-low temperature freezer at -80°C for storage.

[0066] Depend on Figure 3 It can be seen that ( Figure 3In the diagram, black arrows indicate cell infiltration, yellow arrows point to fat vacuoles, and blue arrows indicate cell necrosis. After HE staining, the cell nuclei appear blue and the cytoplasm appears red, allowing for clear observation of liver tissue morphology and pathological damage. In the blank control group (CON), the liver tissue structure of mice was intact, with neatly arranged liver plates in a regular radial distribution, normal hepatocyte morphology, and clear hepatic sinusoidal structure, without obvious fat vacuoles, inflammatory infiltration, or cell necrosis. In the alcohol model group (ETH), the liver tissue of mice showed significant pathological changes, with loose and lightly stained hepatocyte cytoplasm, disordered arrangement of liver plates, and compression and deformation of hepatic sinusoids. Microscopically, numerous fat vacuoles and obvious focal infiltration of inflammatory cells were observed, accompanied by extensive hepatocyte necrosis, severely disrupting the normal physiological structure of the liver, indicating that ethanol-induced severe pathological damage to liver tissue. After intervention with Bacillus subtilis JQ-14 (JQ-14), the number of fat vacuoles in mice was significantly reduced, the degree of inflammatory cell infiltration was greatly alleviated, the lesions of hepatocyte necrosis were significantly reduced, and the overall liver tissue structure was significantly repaired. This indicates that Bacillus subtilis JQ-14 can effectively improve alcohol-induced hepatic steatosis, inflammatory infiltration, and hepatocellular necrosis, and has a good histomorphological protective effect against alcoholic liver injury.

[0067] Depend on Figure 4 It can be seen that in the blank control group (CON) mice, the colonic crypts were neatly and densely arranged, the mucosal epithelium was intact and continuous, there was no obvious inflammatory cell infiltration in the lamina propria, and the intestinal mucosal barrier structure was intact. In the alcohol model group (ETH) mice, after alcohol intervention, the colonic crypts were disordered, the glands were atrophied and deformed, the intestinal epithelial cells were sloughed off and damaged, and a large number of inflammatory cells infiltrated the lamina propria, and the colonic physical barrier was significantly damaged. In contrast, the Bacillus subtilis treatment intervention group (JQ-14) could significantly alleviate ethanol-induced colonic mucosal damage, orderly restore the normal structure of the colonic crypts, repair the damaged intestinal epithelium, reduce mucosal inflammatory infiltration, and effectively maintain the integrity of the colonic mucosal barrier.

[0068] Blood was collected from the orbital sinus of mice to separate serum for the detection of liver injury markers: alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and antioxidant markers: malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). All indicators were detected using Jiangsu Adison reagent kits, and the procedures were performed according to the kit instructions. The test results are shown in Table 4.

[0069] Table 4 As shown in Table 4, after intervention with JQ-14, the levels of ALT and AST were significantly lower than those in the alcohol model group, indicating that JQ-14 can significantly inhibit hepatocyte rupture and leakage, reduce liver damage, and achieve the downregulation of liver function indicators by upregulating the body's antioxidant capacity and reducing hepatocyte oxidative necrosis. At the same time, compared with the alcohol model group, the MDA level in the treatment intervention group was significantly lower and approached that of the blank control group, while SOD and GSH-Px were also significantly increased. This indicates that JQ-14 can effectively alleviate alcohol-induced liver oxidative damage, regulate the body's antioxidant pathways, enhance the expression of endogenous antioxidant enzymes, and inhibit lipid peroxidation, which corresponds to the metabolic activity and structural characteristics of the strain.

[0070] Total microbial DNA was extracted from mouse fecal contents stored at -80 °C, and high-throughput sequencing was performed based on the V3-V4 region of the 16S rRNA gene. Subsequent bioinformatics analysis was used to calculate the gut microbiota α diversity index (e.g., ...). Figure 5 As shown), the relative abundance composition of gut microbiota at the phylum, genus, and species levels (as shown in the figure) Figures 6-8 (As shown in the figure) to investigate the regulatory effect of JQ-14 on alcohol-induced intestinal flora dysbiosis.

[0071] Depend on Figure 5 It can be seen that, compared with the blank control group, the α diversity of the gut microbiota in the alcohol model group was significantly reduced (species richness, community evenness and phylogenetic diversity were all reduced), and the gut microecology was damaged. However, after the treatment intervention group was supplemented with JQ-14, all α diversity indices rebounded significantly, indicating that strain JQ-14 can effectively repair the imbalance of gut microbiota diversity caused by alcohol damage and improve the gut microecological environment.

[0072] Depend on Figure 6 The stacked bar chart of species composition at the phylum level showed that, compared with the CON group, the relative abundance of Firmicutes was significantly decreased and the abundance of Bacteroidetes was significantly increased in the ETH model group, resulting in a decreased Firmicutes / Bacteroidetes (F / B) ratio. Simultaneously, the abundance of pro-inflammatory Proteobacteria was significantly upregulated in the model group, suggesting that alcohol exposure disrupts the dominant gut microbiota structure and induces low-grade inflammatory dysbiosis. Figure 7 The stacked bar chart of species composition at the genus level shows that alcohol exposure significantly decreased the abundance of beneficial bacteria such as *Lactobacillus* and *Bifidobacillus* in the ETH model group, while *Ligilactobacillus* and *Alistipes*, inflammation-related bacteria, were abnormally enriched. Figure 8 The results of the stacked bar chart of species composition at the microbial level show that alcohol significantly inhibits the colonization of protective probiotics such as Lactobacillus acidophilus and induces the specific proliferation of two types of pro-inflammatory bacteria, Lactobacillus murineis and Alternaria masei, demonstrating that long-term alcohol intake can gradually destroy the structure of beneficial intestinal flora and induce intestinal microecological dysbiosis and low-grade inflammatory response.

[0073] In summary, alcohol-induced modeling comprehensively disrupts the gut microbiota structure of mice at the phylum, genus, and species levels: reducing the abundance of beneficial microorganisms such as Firmicutes, Lactobacillus, and Lactobacillus acidophilus, while increasing the abundance of Bacteroidetes and various opportunistic pathogens, thus disrupting the gut microecological balance. However, gavage administration of JQ-14 significantly reversed alcohol-induced gut microbiota dysbiosis: upregulating the Firmicutes / Bacteroidetes ratio, enriching core probiotics such as Lactobacillus and its subgenus Lactobacillus acidophilus, and gradually restoring gut microecological homeostasis. This also indicates that gut microbiota regulation is an important target of JQ-14 in alleviating alcoholic liver injury (gut-liver axis mechanism).

[0074] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis JQ-14, characterized in that, It was deposited on May 22, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38830, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial inoculant, characterized in that, Includes Bacillus subtilis JQ-14 as described in claim 1.

3. A hangover remedy product, characterized in that, Includes Bacillus subtilis JQ-14 as described in claim 1 or the microbial agent as described in claim 2.

4. A liver-protecting product, characterized in that, Includes Bacillus subtilis JQ-14 as described in claim 1 or the microbial agent as described in claim 2.

5. A product for regulating intestinal microbiota, characterized in that, Includes Bacillus subtilis JQ-14 as described in claim 1 or the microbial agent as described in claim 2.

6. The use of Bacillus subtilis JQ-14 as described in claim 1 in the preparation of products that enhance ADH activity and reduce alcohol concentration.

7. The use of Bacillus subtilis JQ-14 as described in claim 1 in the preparation of products that reduce ALT, AST and MDA and increase SOD and GSH-Px enzyme activity.

8. The use of Bacillus subtilis JQ-14 as described in claim 1 in the preparation of products that regulate intestinal microbiota.

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