Human lactobacillus sake TG036 and its application in preparation of beta-nicotinamide mononucleotide

Abstract

This invention provides *Lactobacillus thuringiensis* TG036, a human-derived strain of sake bacteria, and its application in the preparation of β-nicotinamide mononucleotide (NMN). It belongs to the field of microbial technology and has the accession number CCTCC NO: M 20252663. Its 16S rDNA sequence is shown in SEQ ID NO.1. This bacterium combines the functions of synthesizing NMN and degrading uric acid precursors, thus not only combating aging but also eliminating the potential risk of elevated uric acid levels.

Description

Technical Field

[0001] This invention relates to a human-derived Lactobacillus TG036 from sake brewing and its application in the preparation of β-nicotinamide mononucleotide, belonging to the field of microbial technology. Background Technology

[0002] Currently, β-nicotinamide mononucleotide (NMN) is a derivative of nicotinamide adenine dinucleotide (NAD). + NMN is a key precursor in the synthesis of NMN, playing a vital role in physiological processes such as cellular energy metabolism, DNA repair, and anti-aging. With the increasing demand for healthy aging, the market application prospects of NMN are broad. Currently, its preparation methods mainly include chemical synthesis, enzymatic catalysis, and microbial fermentation. Chemical synthesis is cumbersome and may involve toxic reagents; enzymatic methods are costly, limiting large-scale application; microbial fermentation, due to its green and safe characteristics, is gradually becoming a research focus.

[0003] However, existing microbial fermentation methods for NMN production still face significant challenges. On the one hand, natural strains with efficient NMN synthesis capabilities are scarce, and existing strains generally have low yields, making it difficult to meet commercial demands. On the other hand, some strains may accumulate or lack the ability to degrade purine substances (such as adenine and guanosine) during metabolism, and these substances are precursors to uric acid. For the target population of NMN (especially the middle-aged and elderly), they may already have a uric acid metabolic burden. If the probiotic preparations used cannot effectively control these precursor substances, it may potentially increase the risk of hyperuricemia or gout, thus greatly limiting its practical application.

[0004] Therefore, there is an urgent need in current technology to discover a new type of microbial resource that combines the ability to synthesize NMN efficiently with the risk of low uric acid metabolism, in order to provide NMN probiotic preparations with greater advantages in terms of efficacy and safety. Summary of the Invention

[0005] This invention provides a human-derived Lactobacillus TG036 for sake brewing and its application in the preparation of β-nicotinamide mononucleotide, which can effectively solve the above-mentioned problems.

[0006] This invention provides a human-derived Lactobacillus widely distributed in sake ( Latilactobacillus sakei TG036, with accession number CCTCC NO: M 20252663.

[0007] In some embodiments, the 16S rDNA sequence of the above-mentioned human Lactobacillus sakei TG036 is shown in SEQ ID NO.1.

[0008] This invention provides the application of the above-mentioned human-derived Lactobacillus TG036 in the preparation of synthetic β-nicotinamide mononucleotide products.

[0009] This invention provides the application of the above-mentioned human-derived Lactobacillus TG036 in the preparation of a product containing a precursor substance for lowering uric acid.

[0010] In some embodiments, the uric acid precursor includes one or more of adenine, guanosine, inosine, and adenosine.

[0011] The present invention provides a probiotic composition containing the above-mentioned human Lactobacillus sakei TG036 as an active ingredient.

[0012] In some embodiments, the dosage form of the composition includes liquid, powder, granules, capsules, or tablets.

[0013] In some embodiments, the composition is a functional food, dietary supplement, or pharmaceutical preparation.

[0014] The beneficial effects of this invention are:

[0015] The human-derived Lactobacillus TG036 of this invention combines the functions of synthesizing NMN and degrading uric acid precursors, which not only has anti-aging effects but also eliminates the potential risk of elevated uric acid levels.

[0016] The human-derived Lactobacillus TG036 of this invention is derived from humans and is non-GMO. It is sensitive to a variety of commonly used antibiotics such as clarithromycin and penicillin G, and has high safety.

[0017] The NMN produced by the human-derived Lactobacillus TG036 of this invention has two forms: intracellular storage (slow release) and extracellular secretion (direct absorption), resulting in a longer-lasting effect and superior bioavailability.

[0018] The human-derived Lactobacillus TG036 of this invention exhibits good hydrophobicity and self-coagulation ability that increases over time (self-coagulation rate reaches 60.10% after 6 hours), strong colonization potential, and is beneficial for intestinal colonization. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, 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 the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 Morphological photograph of Lactobacillus TG036, a human-derived sake bacterium.

[0021] Figure 2 The phylogenetic tree of *Lactobacillus thuringiensis* TG036, a strain of human sake.

[0022] Figure 3 The chromatograms of adenine and guanosine standards are shown in the embodiments of the present invention.

[0023] Figure 4 Chromatograms of Lactobacillus TG036, a human-derived sake bacterium, degrading adenine and guanosine.

[0024] Figure 5 The chromatograms of inosine and adenosine standards are shown in the embodiments of the present invention.

[0025] Figure 6 Chromatograms of Lactobacillus TG036, a human-derived sake bacterium, degrading inosine and adenosine. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention 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 the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0027] Example 1: Strain Screening

[0028] 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.

[0029] Sequencing results, analyzed by BLAST comparison with the NCBI database, confirmed the strain as *Lactobacillus sakebi* (Sake Lactobacillus). Latilactobacillus sakei It was named Human-derived Sake Lactobacillus TG036. Figure 1 Morphological photograph of Lactobacillus TG036, a human-derived sake bacterium. Figure 2 This is the phylogenetic tree of *Lactobacillus thuringiensis* TG036, a strain of which was deposited in the China Center for Type Culture Collection (CCTCC) in November 2025, with accession number CCTCC NO: M 20252663, located at Wuhan University, Wuhan, China, on November 24, 2025.

[0030] The 16S rDNA sequence of *Lactobacillus TG036*, a human-derived sake bacteria, is as follows:

[0031]

[0032] Example 2 Antibiotic Sensitivity

[0033] The antibiotic susceptibility of human-derived *Lactobacillus thuringiensis* TG036 was assessed using the disk diffusion method. 100 μL of bacterial suspension (approximately 10⁻⁶ g / L) 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.

[0034] As shown in Table 1, human-derived Lactobacillus TG036 is sensitive to clarithromycin, nitrofurantoin, chloramphenicol, spectinomycin, minocycline, and penicillin G; moderately sensitive to clindamycin, erythromycin, and oxacillin; and resistant to tetracycline.

[0035] Table 1. Size of inhibition zone and drug susceptibility results of *Lactobacillus fasciatus* TG036 from human sake.

[0036]

[0037] Example 3: Hydrophobicity of Human-Derived Lactobacillus TG036 in Sake

[0038] Human-derived *Lactobacillus globosum* TG036 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.

[0039]

[0040] In the formula: A0 is the initial absorbance value; A1 is the absorbance value of the bacterial solution after mixing with the hydrophobic agent.

[0041] 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 results in Table 2, human-derived Lactobacillus TG036 in sake brewing exhibited weak hydrophobicity in chloroform, xylene, and ethyl acetate systems (judgment criteria: hydrophobicity <35% is weak, 36%-70% is moderate, and 71%-100% is strong).

[0042] Table 2. Hydrophobicity results of human-derived Lactobacillus TG036 in sake.

[0043]

[0044] Example 4: Self-cohesion of human-derived Lactobacillus TG036 in sake

[0045] The self-aggregation property of *Lactobacillus thuringiensis* TG036 from human sake was 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 .

[0046]

[0047] In the formula: A0 is the initial absorbance value; A t The absorbance values ​​of the bacterial culture are shown at 3 and 6 h after incubation.

[0048] The self-aggregation ability of bacterial strains is positively correlated with their adhesion ability, and is a key indicator for assessing their probiotic potential. As shown in Table 3, the self-aggregation ability of human-derived *Lactobacillus sacchariformis* TG036 increased with prolonged standing time. After 3 hours of standing, the self-aggregation rate of TG036 was 20.24%; after 6 hours of standing, the self-aggregation rate increased to 60.10%. The results indicate that this strain possesses good self-aggregation characteristics.

[0049] Table 3. Hydrophobicity results of human-derived Lactobacillus TG036 in sake.

[0050]

[0051] Example 5: Determination of β-nicotinamide mononucleotide content produced by human-derived *Lactobacillus globosum* TG036 using chemiluminescence method

[0052] The glycerol tubes containing the bacterial strain were removed from the -80℃ freezer and activated by streaking onto MRS agar plates using a disposable inoculation loop. The plates were then incubated upside down at 37℃ under anaerobic conditions for 24 hours. Single colonies were then picked and inoculated into 5 mL of MRS liquid medium, and incubated anaerobicly at 37℃ for 24 hours to obtain the seed culture. The absorbance (OD) of the seed culture at 600 nm was measured. 600Adjust the pH to between 0.8 and 1.0, and transfer 100 μL (2% inoculum, V / V) to 5 mL of MRS liquid medium. Mix well and incubate at 37°C for 24 hours to obtain the working bacterial culture. Centrifuge the working bacterial culture at 5000 rpm for 5 minutes and collect the supernatant for the determination of extracellular NMN content. Resuspend the bacterial precipitate in 5 mL of ultrapure water, centrifuge again, discard the supernatant, then add 2 mL of ultrapure water to resuspend, sonicate (240 W, 20 minutes), centrifuge, and collect the supernatant as the cell-free extract for the determination of intracellular NMN content of the strain.

[0053] Take 70 μL of sample (including the test sample and the standard sample), add 28 μL of 20% acetophenone solution and 28 μL of 2M KOH solution, and mix in a 1.5 mL centrifuge tube. After incubating on ice for 2 minutes, add 126 μL of 88% formic acid, and then incubate at 37℃ for 10 minutes. After the reaction is complete, transfer the sample to a black 96-well plate and measure the fluorescence intensity using a microplate reader at an excitation wavelength of 382 nm and an emission wavelength of 445 nm. Dilute the 200 mM NMN standard solution sequentially to prepare standard samples of 100, 50, 25, 12.5, 6.25, 3.125, and 0 μM, and take 70 μL of each standard sample to measure the fluorescence intensity using the above method. Plot a standard curve with NMN concentration on the x-axis and fluorescence value on the y-axis and fit a linear regression equation.

[0054] The standard curve for determining extracellular NMN content was y = 27866x + 8150.7 (R² = 0.9993), and the standard curve for determining intracellular NMN content was y = 35472x – 19422 (R² = 0.9993). Both standard curves showed a good linear relationship, indicating that the detection method is reliable.

[0055] Fluorescence values ​​measured from the supernatant and cell-free extract of human-derived *Lactobacillus sakei* strain TG036 were substituted into the corresponding standard curves to calculate the NMN content, and the results are listed in Table 4. The data show that the intracellular and extracellular NMN contents of human-derived *Lactobacillus sakei* strain TG036 were 12.67 μM and 1.84 μM, respectively. These results indicate that this strain of human-derived *Lactobacillus sakei* possesses the ability to synthesize NMN, and its NMN is mainly distributed intracellularly.

[0056] Table 4. Extracellular NMN content of *Lactobacillus thuringiensis* TG036 from human sake.

[0057]

[0058] Example 6: Evaluation of the degradation capacity of human-derived *Lactobacillus thuringiensis* TG036 on uric acid precursors.

[0059] The seed culture of the activated first-generation strain was transferred to 40 mL of MRS liquid medium at an inoculation rate of 2% (v / v) and anaerobically cultured at 37 °C for 24 hours. After the culture was completed, the bacterial cells were collected by centrifugation at 5000 rpm for 8 minutes. The bacterial precipitate was resuspended in 5 mL PBS and washed by centrifugation. This process was repeated twice. Finally, the bacterial cells were resuspended in 4 mL PBS to prepare a homogeneous bacterial suspension. 750 μL of the bacterial suspension was placed in a 2 mL EP tube, and an equal volume (750 μL) of substrate solution with a concentration of 400 μg / mL (adenine-guanosine or inosine-adenosine mixed solution) was added. The mixture was co-cultured at 37 °C and 200 rpm for 6 hours on a shaker. After the reaction was completed, the sample was heated in a 95 °C water bath for 10 minutes to terminate the reaction. Subsequently, 1.0 mL of the treatment solution was drawn using a disposable syringe and filtered through a 0.22 μm filter membrane. The filtrate was used for high-performance liquid chromatography (HPLC) analysis.

[0060] The degradation rate is calculated using the following formula:

[0061] α(%) = (1–C / C0) × 100

[0062] Where α represents the degradation rate; C0 is the initial concentration of the standard solution (μg / mL); and C is the concentration of the remaining standard solution after the reaction (μg / mL).

[0063] Quantitative analysis was performed using the external standard method: First, the retention times of adenine, guanosine, inosine, and adenosine standards were determined, and standard curves were plotted using standard solutions of different concentrations. Then, based on the liquid chromatograms of the samples and the standard curves, the specific degradation rates of the four uric acid precursors by each strain were calculated.

[0064] The retention times of adenine, guanosine, inosine, and adenosine were determined using the external standard method, and standard curves were prepared based on different concentrations and retention times. Then, based on the liquid chromatograms and standard curves, the degradation rates of adenine, guanosine, inosine, and adenosine by human-derived Lactobacillus TG036 in sake were calculated.

[0065] Chromatograms of adenine and guanosine standards are shown below. Figure 3 As shown. The chromatogram of human-derived Lactobacillus TG036 degrading adenine and guanosine is shown below. Figure 4 As shown. Chromatograms of inosine and adenosine standards are shown below. Figure 5 As shown. The chromatogram of *Lactobacillus thuringiensis* TG036 degrading inosine and adenosine in human sake is shown below. Figure 6 As shown.

[0066] High-performance liquid chromatography (HPLC) conditions: Waters HPLC system, Agilent ZORBAX SB reversed-phase column. The C18 column was 4.6 × 250 mm in diameter, with a mobile phase of potassium dihydrogen phosphate:methanol = 100:1 (adjusted to pH 3.5 with 5 M phosphoric acid), a flow rate of 1 mL / min, a column temperature of 40 ℃, a retention time of 30 min, and a measurement wavelength of 254 nm.

[0067] The test results are shown in Table 5. As can be seen from Table 5, the degradation rates of the corresponding substrates by the human-derived Lactobacillus TG036 of sake in this invention were 19.64%, 100.00%, 97.64% and 99.31% under the same conditions.

[0068] Table 5. Degradation effects of human-derived Lactobacillus TG036 on adenine, guanosine, inosine, and adenosine.

[0069]

[0070] Example 7: Resistance to Simulated Gastric Fluid

[0071] Activated human-derived *Lactobacillus globosum* TG036 was mixed uniformly with artificial gastric fluid at a 1:1 ratio and incubated in an anaerobic incubator at 37°C for 2 hours to simulate the gastric digestion process. Samples were collected at 0 hours and at the end of the experiment for plate colony counting.

[0072]

[0073] In the formula: A represents gastric juice tolerance (%); N1 and N2 are the initial total viable bacteria count (CFU / mL) of the two parallel experiments; N′1 and N′2 are the total viable bacteria count (CFU / mL) after the two parallel experiments were treated with simulated gastric juice; 2 is the dilution factor conversion factor. Take the average of two parallel data sets.

[0074] The survival rate of human-derived Lactobacillus TG036 in gastric juice at pH 2.0 was 24.47%.

[0075] Example 8: Evaluation of bile salt tolerance

[0076] Human-derived *Lactobacillus globosum* TG036 was inoculated at a rate of 1% into MRS medium containing 0.1% bovine bile salts, with a medium containing no bile salts (0%) serving as a control. The medium was incubated in an anaerobic incubator at 37°C for 2 hours, and samples were taken for viable cell counting. The viable cell counting method was the plate count method, which involved serially diluting the bacterial suspension 10-fold and spreading it onto solid agar plates, anaerobically incubating at 37°C for 24 hours, and then calculating the colony count to determine the viable cell count in the suspension.

[0077] Survival rate (%) = (Number of viable bacteria in the treatment group / Number of viable bacteria in the control group) × 100%

[0078] When the bile salt content in the culture medium was 0.1%, the survival rate of human-derived Lactobacillus TG036 in sake was 23.68%.

[0079] The human-derived *Lactobacillus thuringiensis* TG036 provided by this invention possesses outstanding substantive features and significant progress. Its inventiveness is mainly reflected in the unexpected technical effects of "dual-effect synergy" and "metabolic blockade":

[0080] 1. This invention resolves the technical contradiction between NMN biosynthesis and the burden of exogenous purine metabolism.

[0081] In existing technologies, NMN supplementation through microbial fermentation often faces limitations in terms of specificity. Furthermore, some highly metabolically active strains may accumulate purine metabolic byproducts during the synthesis of nucleotides, posing a risk of inducing hyperuricemia in the elderly or those susceptible to gout. The TG036 strain of this invention, while possessing a stable β-nicotinamide mononucleotide (NMN) synthesis capacity (4.23 mg / L intracellularly, 0.61 mg / L extracellularly), exhibits extremely high efficiency in clearing purine-induced uric acid precursors. Specifically, the strain achieves a 100.00% degradation rate of guanosine, a 99.31% degradation rate of adenosine, and a 97.64% degradation rate of inosine. This metabolic characteristic of "simultaneously producing beneficial factors (NMN) and efficiently clearing harmful precursors (purine nucleosides)" is uncommon among similar lactobacilli, successfully establishing a balance between supplementing cellular energy factors and maintaining uric acid homeostasis, overcoming the technical shortcomings of existing probiotic preparations that are either functionally limited or have metabolic side effects.

[0082] 2. Excellent targeted colonization potential and bioavailability

[0083] Unlike traditional industrial fermentation strains that only pursue yield, the innovativeness of this invention's strain, as a colonizing probiotic, lies in its unique surface characteristics. TG036 exhibits a significantly enhanced self-aggregation ability over time (jumping from 20.24% at 3 hours to 60.10% at 6 hours). This high self-aggregation is a key indicator for probiotic colonization in the intestinal mucosa. Combined with its NMN secretion characteristics (intramural storage and sustained release + extracellular secretion), TG036 can colonize in the intestine and continuously and dynamically provide NMN. This "in-situ production + sustained-release absorption" administration mode theoretically has higher bioavailability and longer-lasting effects compared to orally administered, easily degraded pure NMN, constituting a significant advancement in the formulation application of this invention.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A type of human-derived widely distributed lactobacillus in sake ( Latilactobacillus sakei TG036, characterized in that, Its accession number is CCTCC NO: M 20252663.

2. The human-derived *Lactobacillus TG036* 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 TG036 of sake brewing as described in claim 1 in the preparation of synthetic β-nicotinamide mononucleotide products.

4. The application of the human-derived *Lactobacillus thuringiensis* TG036 from sake brewing as described in claim 1 in the preparation of a product containing uric acid-lowering precursors, characterized in that... The uric acid precursor is one or more of adenine, guanosine, inosine, and adenosine.

5. A probiotic composition, characterized in that, The composition contains the human-derived Lactobacillus TG036 of claim 1 as an active ingredient.

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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