Human lactobacillus sake TG035 and its application in preparing antioxidant and acetaldehyde dehydrogenase product

Abstract

This invention discloses a human-derived *Lactobacillus TG035* for sake brewing and its application in antioxidant and acetaldehyde dehydrogenase-producing products, belonging to the field of microbial technology. The strain TG035 has the preservation number CCTCC NO: M 20252662, and its 16S rDNA sequence is shown in SEQ ID NO.1. This strain possesses the dual function of producing acetaldehyde dehydrogenase and superoxide dismutase.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a human-derived Lactobacillus TG035 of sake brewing and its applications. Background Technology

[0002] According to the guidelines of the International Agency for Research on Cancer (IARC), alcohol is classified as a Group 1 human carcinogen, indicating a clear link to the development of human cancer. Excessive alcohol metabolism increases the liver's oxygen consumption and produces excessive reactive oxygen species (ROS), inducing oxidative stress and inflammatory responses, thus damaging the liver. Furthermore, ROS activate inflammatory pathways, triggering inflammatory responses and further leading to liver damage. Acetaldehyde, a toxic intermediate product of alcohol metabolism, can form adducts with cellular DNA and proteins, exacerbating oxidative stress and thus impairing hepatocyte function. Moreover, long-term alcohol consumption leads to gut microbiota dysbiosis, thereby damaging the intestinal mucosal barrier and inducing inflammation, ultimately resulting in alcoholic liver disease and cirrhosis.

[0003] Acetaldehyde dehydrogenase (ALDH) is an important enzyme primarily located in the liver, widely found in human and animal livers, plant and microbial cells. It protects the human body from the harmful effects of acetaldehyde by converting it into harmless acetic acid. Currently, ALDH is mainly extracted from animal liver and pancreatic cells; however, the limited availability and difficulty of extraction result in high costs.

[0004] In recent years, the close link between gut microbiota disorders and liver damage has been increasingly emphasized. The bidirectional interaction between the gut and liver, the "gut-liver" axis, has become a key focus of liver disease research. As an important component of the gut microbiota, probiotics have shown considerable potential in improving alcoholic liver disease. These probiotics can reduce host ethanol absorption by enhancing the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) or enhance the liver's antioxidant capacity by increasing superoxide dismutase (SOD) and glutathione (GSH) levels, thereby mitigating inflammatory and oxidative damage pathways.

[0005] Existing methods for producing ALDH and SOD include:

[0006] (1) Traditional separate extraction and compounding: ALDH and SOD are extracted separately from different microbial sources and then compounded as needed. The technology is relatively mature and intuitive, and is suitable for obtaining enzymes from natural sources. However, this method lacks a high-throughput screening method for high-quality mutants, which may result in enzymes with low activity and low excretion efficiency.

[0007] (2) Single-strain co-expression: The genes for ALDH and SOD are simultaneously introduced into the same engineered bacterium (such as Escherichia coli or Bacillus subtilis). However, the vector construction for this method is complex, and the two enzymes may compete for resources within the bacterium, leading to an imbalance in expression. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a human-derived Lactobacillus TG035 for sake brewing and its application in the preparation of antioxidant and acetaldehyde dehydrogenase-producing products. The strain has the dual function of producing acetaldehyde dehydrogenase and superoxide dismutase, and can be used to develop safe, effective, and multi-beneficial microbial preparations.

[0009] The technical solution of the present invention is as follows:

[0010] This invention provides a human-derived Lactobacillus widely distributed in sake ( Latilactobacillus sakei TG035, strain TG035 has the accession number CCTCC NO: M 20252662.

[0011] In some embodiments, the 16S rDNA sequence of the human-derived *Lactobacillus thuringiensis* TG035 is shown in SEQ ID NO. 1.

[0012] This invention provides the application of the above-mentioned human-derived Lactobacillus TG035 in the preparation of synthetic acetaldehyde dehydrogenase products.

[0013] This invention provides the application of the above-mentioned human-derived Lactobacillus TG035 in the preparation of superoxide dismutase products.

[0014] This invention provides the application of the above-mentioned human-derived Lactobacillus TG035 in the preparation of probiotic preparations with functions of relieving hangovers, protecting the liver, or antioxidation.

[0015] In some embodiments, the probiotic preparation reduces liver oxidative stress damage by producing acetaldehyde dehydrogenase to degrade acetaldehyde and by producing superoxide dismutase to scavenge free radicals.

[0016] The present invention provides a probiotic composition containing the above-mentioned human Lactobacillus sakei TG035 as an active ingredient.

[0017] In some embodiments, the dosage form of the composition includes liquid, powder, granules, capsules, or tablets.

[0018] In some embodiments, the composition is a functional food, dietary supplement, or pharmaceutical preparation.

[0019] The beneficial effects of this invention are:

[0020] The human-derived Lactobacillus TG035 of this invention is derived from healthy human samples, is non-GMO, and has excellent human adaptability.

[0021] The human-derived Lactobacillus TG035 of this invention combines acetaldehyde degradation (ALDH) and antioxidant balancing (SOD) functions for the first time, which can simultaneously reduce acetaldehyde toxicity and oxidative damage caused by alcohol.

[0022] The human-derived Lactobacillus TG035 of this invention possesses excellent self-aggregation and hydrophobicity, and strong colonization ability, which is beneficial for colonization and function in the intestine. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Morphological diagram of Lactobacillus TG035 strain from human sake.

[0025] Figure 2 Phylogenetic tree of Lactobacillus TG035, a human-derived sake bacterium.

[0026] Figure 3 A standard curve for determining the total protein content of Lactobacillus TG035 in human-derived sake. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure. Therefore, the following detailed description of the embodiments of this disclosure provided in the accompanying drawings is not intended to limit the scope of the claimed disclosure, but merely represents selected embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure.

[0028] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0029] Example 1: Strain Screening

[0030] Donor fecal samples were collected from healthy adults in China. First, appropriately diluted fecal samples were spread onto MRS solid medium and incubated in a 37°C anaerobic incubator. Then, single colonies from the medium were streaked for purification, and the purified colonies were inoculated into MRS liquid medium for amplification. Subsequently, genomic DNA was extracted from the bacterial strains using a rapid bacterial genomic DNA extraction kit. The extracted DNA was used as a template for PCR amplification, and the amplified products were finally sent for sequencing.

[0031] Discussion of Results:

[0032] Sequencing results, analyzed by BLAST comparison with the NCBI database, confirmed that the strain is *Lactobacillus sakebifolia* (Sakebifolia). Latilactobacillus sakei It was named Human-derived Sake Lactobacillus TG035. Figure 1 Morphological diagram of Lactobacillus sakei TG035 strain; Figure 2 Lactobacillus sakei TG035 ( Latilactobacillus sakei The phylogenetic tree of the strain is shown. The strain was deposited at the China Center for Type Culture Collection (CCTCC) in November 2025, with accession number CCTCC NO: M20252662, located at Wuhan University, Wuhan, China, on November 24, 2025.

[0033] The 16S rDNA sequence of *Lactobacillus TG035*, a human-derived sake bacteria, is as follows:

[0034]

[0035] Example 2 Antibiotic Sensitivity

[0036] The antibiotic susceptibility of human-derived *Lactobacillus thuringiensis* TG035 was assessed using the disk diffusion method. 100 μL of bacterial suspension (approximately 10⁻⁶ oz.) was used. 8 The CFU / mL concentration was evenly spread onto MRS agar plates. Test strips containing different antibiotics (including clarithromycin, nitrofurantoin, chloramphenicol, clindamycin, tetracycline, erythromycin, spectinomycin, minocycline, oxacillin, and penicillin G) were placed on the plate surface, with antibiotic-free strips used as blank controls. After incubating the plates anaerobically at 37°C for 48 h, the diameter of the inhibition zone was measured using calipers, and the results were interpreted according to the CLSI Antimicrobial Susceptibility Testing Standards.

[0037] Discussion of Results:

[0038] As shown in Table 1, human-derived Lactobacillus TG035 is sensitive to clarithromycin, nitrofurantoin, chloramphenicol, clindamycin, minocycline, and penicillin G; moderately sensitive to tetracycline, erythromycin, and oxacillin; and resistant to spectinomycin.

[0039] Table 1. Size of inhibition zone and drug susceptibility results of *Lactobacillus fasciatus* TG035 from human sake.

[0040]

[0041] Example 3: Hydrophobicity of Human-Derived Lactobacillus TG035 in Sake

[0042] Human-derived *Lactobacillus globosum* TG035 bacterial suspension was centrifuged, and the bacterial cells were collected and washed twice with PBS. Using PBS as a blank control, the bacterial suspension was adjusted to an OD value of approximately 0.80 at 600 nm, and its initial absorbance was measured and recorded as A0. 3 mL of the adjusted bacterial suspension was taken, and 1 mL of a hydrophobic agent (including xylene, ethyl acetate, or chloroform) was added. After standing for 5 min, the suspension was vortexed for 2 min, and then allowed to stand for another 30 min to allow for layering. The absorbance of the aqueous phase was measured and recorded as A1.

[0043]

[0044] In the formula: A0 is the initial absorbance value; A1 is the absorbance value of the bacterial solution after mixing with the hydrophobic agent.

[0045] Discussion of Results:

[0046] The beneficial effects of probiotics, such as eliminating pathogens, adhering to the intestinal epithelium, and colonizing the gastrointestinal tract, all depend on the hydrophobicity of their cell surface. According to the test results in Table 2, human-derived Lactobacillus TG035 in chloroform, xylene, and ethyl acetate systems all exhibited weak hydrophobicity (judgment criteria: hydrophobicity <35% is weak, 36%-70% is moderate, and 71%-100% is strong).

[0047] Table 2. Hydrophobicity results of human-derived Lactobacillus TG035 in sake.

[0048]

[0049] Example 4: Self-cohesion of human-derived Lactobacillus TG035 in sake

[0050] The self-aggregation properties of *Lactobacillus globosum* TG035 and TG036 from human-derived sake bacteria were determined. First, the bacterial suspension was centrifuged and the cells collected, then washed twice with sterile physiological saline. Using sterile physiological saline as a reference, the absorbance of the bacterial suspension at 600 nm was adjusted to approximately 1.0, and this initial value A0 was recorded. Subsequently, 8 mL of the bacterial suspension was aliquoted into identical test tubes and incubated under suitable conditions. At the 3rd and 6th hour of incubation, the supernatant bacterial suspension was collected, and its absorbance at 600 nm was measured and recorded as A. t .

[0051]

[0052] In the formula: A0 is the initial absorbance value; At is the absorbance value of the bacterial solution at 3 and 6 h after incubation.

[0053] Discussion of Results:

[0054] The self-aggregation ability of a bacterial strain is positively correlated with its adhesion ability, and is a key indicator for assessing its probiotic potential. As shown in Table 3, the human-derived *Lactobacillus sacchariformis* TG035 exhibited an increased self-aggregation ability with prolonged standing time. After 3 hours of standing, the self-aggregation rate of TG035 was 18.91%; after 6 hours of standing, its self-aggregation rate increased to 85.58%. The results indicate that this strain possesses good self-aggregation characteristics.

[0055] Table 3. Autocoagulation rate results of Lactobacillus TG035 in human-derived sake.

[0056]

[0057] Example 5: Evaluation of the ethanol resistance of human-derived Lactobacillus TG035 (a type of Lactobacillus widely distributed in sake).

[0058] The bacterial strain was removed from the -80 ℃ freezer and activated by streaking onto MRS agar plates using a disposable inoculation loop in a clean bench. After anaerobic incubation at 37 ℃ for 24 h, a single colony was inoculated into 5 mL of MRS liquid medium and incubated anaerobically at 37 ℃ for 24 h to obtain the first-generation bacterial culture. Then, 50 μL (1%, v:v) of the first-generation bacterial culture was inoculated into 5 mL of MRS liquid medium, shaken well, and incubated in an anaerobic incubator at 37 ℃ for 24 h to obtain the second-generation bacterial culture. The OD of the second-generation bacterial culture was measured. 600 Adjust the value to 1.0 to obtain the working bacterial solution, which is then ready for use.

[0059] Liquid culture medium containing different concentrations of ethanol was added to 96-well deep-well plates. Each group was configured with three replicates, and the medium concentration was OD0.05. 600 A working bacterial culture of 1.0 μL was inoculated into deep wells containing 0%, 6%, and 12% ethanol at an initial inoculum of 1% v / v. After incubation at 37°C for 24 h, the absorbance at 600 nm was measured using a microplate reader to assess cell viability.

[0060] Discussion of Results:

[0061] As shown in Table 4, the survival rate of the human-derived Lactobacillus TG035 of the present invention after 24 h of culture in MRS containing 6% ethanol was 0.28±0.12, which was not under ethanol stress, demonstrating good ethanol tolerance.

[0062] Table 4 Survival rate of human-derived Lactobacillus TG035 in sake

[0063]

[0064] Example 6: Determination of total protein content of *Lactobacillus thuringiensis* TG035 in human-derived sake.

[0065] According to the Beyotime BCA protein concentration assay kit instructions, a BCA protein concentration standard curve was prepared. The standard curve was plotted with protein concentration on the x-axis and fluorescence value on the y-axis, and a linear regression equation was fitted.

[0066] Discussion of Results:

[0067] The standard curve is as follows Figure 3 As shown, the BCA protein standard curve is y = 0.7511x + 0.09745 (R²). 2 =0.9965), the standard curve showed a good linear relationship, indicating that the detection method is reliable. The supernatant of the human-derived *Lactobacillus thuringiensis* TG035 strain was lysed and cultured at OD0.05. 562The measured fluorescence values ​​were substituted into the standard curve formula to calculate the protein content of the strain's lysate supernatant. The results are shown in Table 5. The protein content of the cell lysate supernatant of human Lactobacillus TG035 was 0.48 mg / mL.

[0068] Table 5. Protein content in the supernatant of cell lysate of *Lactobacillus thuringiensis* TG035 (human-derived sake bacteria).

[0069]

[0070] Example 7: Determination of acetaldehyde dehydrogenase activity produced by *Lactobacillus thuringiensis* TG035 in human sake.

[0071] A 2% inoculum of the first-generation culture of the ethanol-resistant strain was inoculated into a centrifuge tube containing 50 mL of MRS liquid medium and anaerobic fermented at 37 °C for 24 h to obtain the fermentation broth. 30 mL of the fermentation broth was centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and all bacterial cells were collected. The cells were washed with 10 mL of PBS and resuspended, then ultrasonically disrupted (300 W power, 3 s on, 7 s off, working time 30 min). The cells were then centrifuged at 8000 r / min and 4 °C for 10 min, the precipitate was discarded, and the supernatant was used to determine the enzyme activity.

[0072] Acetaldehyde dehydrogenase activity assay, refer to Giebu The method used by towicz et al. involved Tris-HCl (pH 8.0) and coenzyme NAD. + Acetaldehyde, KCl, β-mercaptoethanol, and ultrapure water were mixed in advance according to the amounts specified in Table 5 of the ALDH activity assay system. After incubating at 35 °C for 10 min, the crude enzyme was added. PBS was used as a blank control. The change in absorbance at 340 nm was quickly detected using a microplate reader. The activity unit was defined as 1 μmol / min of NADH oxidized per milliliter of sample at 25 °C. Absorbance values ​​were read every 1 min, with a total reaction time of 5 min. The ALDH enzyme activity was calculated based on the change in absorbance per minute.

[0073] Table 6 ALDH Activity Assay System

[0074]

[0075] Enzyme activity is defined as a change of 0.001 in absorbance at 340 nm over 1 minute at 35°C.

[0076] The enzyme activity of ALDH is calculated using the formula:

[0077]

[0078] Note: 340 =Measuring tube 340 - Control tube 340

[0079] The test results are shown in Table 7. The crude acetaldehyde dehydrogenase activity of the supernatant of the human-derived Lactobacillus TG035 strain of sake in this invention is 0.48 U / mL, and the acetaldehyde dehydrogenase activity per 1 mg of protein is 1 U.

[0080] Table 7. Acetaldehyde dehydrogenase production capacity of *Lactobacillus thuringiensis* TG035 from human sake brewing.

[0081]

[0082] Example 8: Determination of Superoxide Dismutase (SOD) Activity Produced by *Lactobacillus TG035* in Human Sake Brewery

[0083] The SOD activity of the bacteria was determined according to the instructions of the Beyotime Total SOD Activity Detection Kit. The results are shown in Table 8.

[0084] Superoxide dismutase (SOD) is the most important enzyme for scavenging superoxide anions, and maintaining a certain level of SOD in the body is crucial for maintaining redox balance. The human-derived *Lactobacillus TG035* strain of this invention has a specific SOD activity of 8.83 U / mg in its supernatant, demonstrating a certain antioxidant capacity and the ability to maintain redox balance in the body.

[0085] Table 8. Acetaldehyde dehydrogenase production capacity of *Lactobacillus thuringiensis* TG035 from human sake brewing.

[0086]

[0087] Example 9: Resistance to Simulated Gastric Fluid

[0088] Human-derived *Lactobacillus globosum* TG035 from sake was mixed with artificial gastric juice at a 1:1 ratio and then anaerobically digested at 37°C for 2 hours to simulate the gastric environment. Gastric acid tolerance was assessed by comparing plate colony counts at 0 and 2 hours after treatment.

[0089]

[0090] In the formula: A: gastric juice tolerance (%); N1, N2: initial total number of viable bacteria (CFU / mL) before treatment in two parallel experiments; N′1, N′2: total number of viable bacteria (CFU / mL) after treatment with simulated gastric juice in two parallel experiments; when calculating, the average value of the data from two parallel experiments is taken, and the dilution factor conversion factor 2 is introduced.

[0091] The survival rate of human-derived Lactobacillus TG035 in gastric juice at pH 2.0 was 38.96%.

[0092] Example 8: Evaluation of bile salt tolerance

[0093] To assess the bile salt tolerance of the strain, human-derived *Lactobacillus globosum* TG035 was inoculated at a 1% inoculum into MRS medium containing 0.1% bovine bile salts, with a bile-free (0%) medium serving as a control. All treatments were incubated anaerobically at 37°C for 2 hours before sampling. Viable cell counts were performed using the plate count method: samples were serially diluted 10-fold, plated on solid agar plates, and anaerobically incubated at 37°C for 24 hours before counting to determine the viable cell count (CFU / mL).

[0094] Survival rate (%) = (Number of viable bacteria in the treatment group / Number of viable bacteria in the control group) × 100%

[0095] When the bile salt content in the culture medium was 0.1%, the survival rate of human Lactobacillus TG035 in sake was 27.44%.

[0096] In summary, through extensive screening, this invention unexpectedly discovered that the human-derived *Lactobacillus thuringiensis* TG035 can simultaneously and efficiently produce acetaldehyde dehydrogenase (ALDH) and superoxide dismutase (SOD). The specific enzyme activity of ALDH in its cell lysis supernatant reaches 1.00 U / mg, and the specific enzyme activity of SOD reaches 8.83 U / mg. This characteristic of combining "acetaldehyde toxicity degradation" and "free radical scavenging" within a single natural strain not only simplifies the production process but also creates a natural synergistic effect of "alcohol detoxification and liver protection" in the intestines. Its technical effect is significantly superior to that of simple enzyme preparations.

[0097] As a probiotic for relieving hangovers, it must survive and function in the harsh intestinal environment containing alcohol. Experimental data shows that TG035 can maintain a high survival rate even under 6% high-concentration ethanol stress. In addition, this strain exhibits extremely strong self-aggregation properties, with a self-aggregation rate as high as 85.58% after standing for 6 hours. Combined with its good hydrophobicity, this ensures that the strain has excellent colonization ability in the complex gastrointestinal environment, thereby guaranteeing the continuous and stable expression and function of the biological enzyme in vivo.

[0098] Compared to existing technologies that use genetically engineered single-strain co-expression (such as engineered Escherichia coli or Bacillus subtilis), the TG035 strain of this invention is derived from healthy human samples and is a non-GMO natural probiotic. This not only avoids the potential biosafety risks and metabolic burdens that engineered bacteria may pose, leading to unstable expression, but also overcomes the limitations of high extraction costs and difficulties associated with animal-derived ALDH, opening up a new technological path for the preparation of safe, inexpensive, and highly effective hangover relief and liver protection products.

[0099] The above description is merely a preferred embodiment of this disclosure and is not intended to limit this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A strain of human-derived Lactobacillus widely distributed in sake ( Latilactobacillus sakei TG035, characterized in that, The preservation number of the human-derived Lactobacillus TG035 in sake is CCTCC NO: M 20252662.

2. The human-derived *Lactobacillus TG035* from sake brewing according to claim 1, characterized in that, Its 16S rDNA sequence is shown in SEQ ID NO.

1.

3. The application of the human-derived Lactobacillus TG035 of sake as described in claim 1 in the preparation of synthetic acetaldehyde dehydrogenase products.

4. The application of the human-derived Lactobacillus TG035 of sake as described in claim 1 in the preparation of superoxide dismutase products.

5. A probiotic composition, characterized in that, The composition contains the human-derived Lactobacillus TG035 of sake as the active ingredient as described in claim 1.

6. The probiotic composition according to claim 5, characterized in that, The dosage form of the composition includes liquid, powder, granules, capsules or tablets.

7. The probiotic composition according to claim 5, characterized in that, The composition is a functional food, dietary supplement, or pharmaceutical preparation.

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