Lactobacillus gasseri with high efficiency of utilizing tryptophan to produce indole-3-lactic acid and application thereof

Abstract

The application discloses a lactobacillus gasseri for efficiently producing indole-3-lactic acid from tryptophan and application thereof, and has the characteristics that the strain is named as lactobacillus gasseri, the preservation number is CGMCC No.37546, the strain metabolically produces indole-3-lactic acid after anaerobic fermentation in a culture medium containing tryptophan, and the application of the lactobacillus gasseri in preparing products for relieving constipation, regulating emotional state and improving the microecological balance of female reproductive tract is provided, and the application of the strain in preparing pathogenic bacteria inhibitors and antioxidants is further provided. Lactobacillus gasseri The strain can efficiently produce indole-3-lactic acid from tryptophan, has inhibiting effects on escherichia coli, staphylococcus aureus and gardnerella vaginalis, can eliminate superoxide anions, DPPH free radicals and ABTS free radicals, and has good antioxidant performance.

Description

Technical Field

[0001] This invention relates to Lactobacillus gasseri, and more particularly to a Lactobacillus gasseri that efficiently utilizes tryptophan to produce indole-3-lactic acid and its applications. Background Technology

[0002] Indole derivatives are a class of natural products produced by the metabolism of tryptophan, and indole-3-lactic acid is one such indole derivative. This substance is an endogenous ligand for the aryl hydrocarbon receptor (AhR) and possesses anti-inflammatory effects, playing a significant role in relieving constipation, inflammatory bowel disease, and regulating mood. Current research shows that indole-3-lactic acid can act as a ligand for the aryl hydrocarbon receptor (AhR) in the human gut. AhR activation regulates intestinal mucosal immune homeostasis and epithelial barrier genes, promotes the expression of tight junction proteins, promotes the repair of intestinal epithelial cells, and reduces intestinal permeability; it also inhibits the NF-κB pathway, downregulates inflammatory factors, and reduces intestinal inflammation, all of which play a significant role in relieving constipation and inflammatory bowel disease. Furthermore, AhR activation can significantly inhibit neuroinflammation, reduce the level of pro-inflammatory factors in the brain, thereby protecting neurons in brain regions such as the hippocampus, improving neurogenesis inhibition caused by chronic stress, and improving depressive / anxiety-like behaviors.

[0003] Lactobacillus gasseri ( Lactobacillus gasser Lactobacillus gasseri is considered an effective probiotic for the prevention and treatment of vaginal infections. It can produce lactic acid through the fermentation of sugars, lowering the pH level in the vagina; and it possesses strong antibacterial capabilities by secreting antibacterial substances (such as bacteriocins), which can effectively inhibit the growth of pathogenic bacteria and reduce the risk of vaginal infections. It improves the vaginal microecological environment, inhibits the growth of pathogenic microorganisms, and reduces the incidence of common vaginal infections such as bacterial vaginosis and yeast infections. Currently, there are no research reports on Lactobacillus gasseri that utilizes tryptophan to produce indole-3-lactic acid. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Lactobacillus gasseri that efficiently utilizes tryptophan to produce indole-3-lactic acid and its application.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problems is: a strain of *Lactobacillus gasseri* that efficiently utilizes tryptophan to produce indole-3-lactic acid, and the strain is classified and named *Lactobacillus gasseri* (…). Lactobacillus gasseri The strain QS766, with accession number CGMCC No.37546, was deposited on January 26, 2026. The depository is the China General Microbiological Culture Collection Center, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0006] The present invention also provides the application of the above-mentioned Lactobacillus gasseri in the production of indole-3-lactic acid, wherein the Lactobacillus gasseri strain produces indole-3-lactic acid after anaerobic fermentation in a tryptophan-containing culture medium.

[0007] Furthermore, the tryptophan-containing culture medium is an MRS liquid culture medium containing a final concentration of 1-3 g / L tryptophan and 0.1-1 wt% L-cysteine ​​hydrochloride.

[0008] The present invention also provides the application of the above-mentioned Lactobacillus gasseri in the preparation of products for relieving constipation.

[0009] The present invention also provides the application of the above-mentioned Lactobacillus gasseri in the preparation of products for regulating mood.

[0010] The present invention also provides the application of the above-mentioned Lactobacillus gasseri in the preparation of products that improve the microecological balance of the female reproductive tract.

[0011] The present invention also provides a microbial preparation containing the above-mentioned Lactobacillus gasseri, wherein the microbial preparation is selected from bacterial suspension, fermentation supernatant, lyophilized bacterial powder or a combination thereof.

[0012] This invention also provides the use of the above-mentioned Lactobacillus gasseri or microbial preparation in the preparation of pathogenic bacteria inhibitors, wherein the pathogenic bacteria are selected from Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ) and Gardnerella vaginalis ( Gardnerella vaginalis At least one of the following.

[0013] The present invention also provides the use of the above-mentioned Lactobacillus gasseri or microbial preparations in the preparation of antioxidants.

[0014] Furthermore, the antioxidants include superoxide anion scavengers, ABTS free radical scavengers, and / or DPPH free radical scavengers.

[0015] Compared with existing technologies, the advantages of this invention are as follows: This invention discloses a strain of *Lactobacillus gasseri* that efficiently utilizes tryptophan to produce indole-3-lactic acid (ILA) and its applications. This strain can efficiently utilize tryptophan to metabolize and generate indole-3-lactic acid (ILA). Using MRS medium containing 2 g / L tryptophan and 0.5% L-cysteine ​​hydrochloride for fermentation, the ILA content can reach 90.86 mg / L. Furthermore, this strain exhibits inhibitory effects on pathogenic bacteria such as *Escherichia coli*, *Staphylococcus aureus*, and *Gardnerella vaginalis*. It can scavenge superoxide anions, DPPH free radicals, and ABTS free radicals, demonstrating good antioxidant properties. Under conventional culture conditions, it can produce up to 9.86 g / L of lactic acid, indicating its excellent fermentation acid-producing capacity. Evaluation according to national food safety standards shows that this strain is sensitive to 10 common clinical and food-related antibiotics, including tetracycline, erythromycin, vancomycin, and penicillin, indicating no acquired resistance and high biosafety, making it suitable for the development of food and health-related products.

[0016] In summary, the Lactobacillus gasseri strain QS766 of this invention, which efficiently utilizes tryptophan to produce indole-3-lactic acid, integrates multiple excellent properties such as high efficiency in producing functional metabolites (indole-3-lactic acid and lactic acid), broad-spectrum antibacterial activity, strong antioxidant effect, and high biocompatibility. It has significant application potential in the fields of functional foods, probiotic preparations, antimicrobial agents, and antioxidant product development. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of Lactobacillus gasseri QS766.

[0018] Figure 2 Figure showing the results of the ability of Lactobacillus gasseri QS766 to produce indole-3-lactic acid;

[0019] Figure 3 The standard curve for indole-3-lactic acid;

[0020] Figure 4 The standard curve for lactate;

[0021] Figure 5 Diagrams showing the diameters of inhibition zones of Lactobacillus gasseri QS766 bacterial culture, supernatant, and bacterial suspension against Escherichia coli, Staphylococcus aureus, and Gardnerella vaginalis. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0023] Specific Implementation Example 1: Isolation and Identification of Strains.

[0024] 1. Strains Isolation

[0025] Dissolve 1g of fecal matter from a centenarian in a centrifuge tube containing 9mL of sterile saline solution, vortex to mix, and set aside. Dilute to 10⁻⁶ using a serial dilution method. -6 From 10 respectively -4 10 -5 10 -6 Three gradients of 100 μL were evenly spread onto MRS solid medium supplemented with 0.5 wt% L-cysteine ​​hydrochloride. The culture dishes were placed in an anaerobic workstation at 37°C and incubated for 48 h. Colony morphology was observed and recorded. Single colonies were picked up using an inoculation loop and streaked onto MRS solid medium supplemented with 0.5 wt% L-cysteine ​​hydrochloride. The culture was then anaerobic at 37°C for 48 h to obtain pure colonies. The pure colonies obtained from the plates were inoculated into MRS liquid medium supplemented with 0.5 wt% L-cysteine ​​hydrochloride and anaerobic at 37°C for 16-18 h. The pure strain was then mixed with a 50% glycerol solution in an equal proportion and stored at -80°C. A total of 110 lactic acid bacteria strains were screened from fecal samples of centenarians. Among them, strain QS766 showed significant advantages in growth performance and acid tolerance. Therefore, strain QS766 was identified as the key strain for this experiment.

[0026] 2. Strain identification

[0027] Genomic DNA was extracted from the strain obtained in step 1, and then amplified and sequenced using the universal 16S rRNA primers 27F and 1492R to identify the strain species. The primer sequences of the universal 16S rRNA primers 27F and 1492R are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'; PCR system components: 1 μL DNA template, 1 μL 27F, 1 μL 1492R, 22 μL sterile water, 2×Taq PCR Master Mix (PCR premix).

[0028] The PCR program was as follows: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, for 30 cycles; final extension at 72℃ for 10 min. The amplified PCR product was removed, and 1.5-2 μL of the PCR product was added to an agarose gel. The electrophoresis apparatus voltage was set to 99-100V, and the gel was run for 20-30 min. After the gel was removed, the DNA bands were observed using a gel imaging system, and the target fragment length was determined to be 1500 bp.

[0029] Figure 1 As shown. Based on morphological and 16S rRNA identification, strain QS766 was identified as Lactobacillus gasseri.

[0030] This strain is currently deposited at the China General Microbiological Culture Collection Center and is classified as *Lactobacillus gasseri*. Lactobacillus gasseri The strain QS766, with accession number CGMCC No.37546, was deposited on January 26, 2026. The deposit address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, and the depositary institution is the China General Microbiological Culture Collection Center.

[0031] Specific Example 2: Analysis of the ability of Lactobacillus gasseri QS766 to produce indole-3-lactic acid.

[0032] 1. Preparation of Lactobacillus gasseri seed culture

[0033] Lactobacillus gasseri QS766, stored at -80℃, was taken out and inoculated into MRS liquid medium supplemented with 0.5% L-cysteine ​​hydrochloride at a volume ratio of 2% for 3 generations of activation culture.

[0034] 2. Determination of indole-3-lactic acid, a tryptophan metabolite in Lactobacillus gasseri

[0035] (1) Seed culture was inoculated into 10 mL of MRS liquid medium containing 2 g / L tryptophan and 0.5 wt% L-cysteine ​​hydrochloride at a 2% (v / v) inoculation rate. The culture was anaerobic at 37 °C for 48 h, and repeated 3 times. The culture medium was centrifuged at 8000 rpm / min at 4 °C to collect the supernatant. The supernatant was mixed with methanol pre-cooled at -20 °C at a volume ratio of 1:4 and treated at -20 °C for 30 min. The supernatant was then centrifuged at 12000 rpm / min at 4 °C and nitrogen purging was performed at room temperature to obtain the sample to be tested.

[0036] (2) Take a sample with a concentration of 1 mg / mL, filter it through a 0.22 μm filter, and perform high performance liquid chromatography analysis. The results are as follows: Figure 2 As shown. The conditions for high-performance liquid chromatography (HPLC) analysis included: an Agilent 1260 Infinity HPLC system; a ZORBAX SB-Aq 4.6 x 150 mm 3.5-Micron chromatograph for separation; a wavelength of 280 nm; mobile phases A (0.1% formic acid aqueous solution) and B (100% acetonitrile); an injection volume of 10 μL; and a flow rate of 1 mL / min. The gradient elution program was as follows:

[0037] Table 1 Gradient elution program

[0038]

[0039] The peak area of ​​indole-3-lactic acid at different concentrations was determined using the external standard method. A standard curve was obtained by plotting the indole-3-lactic acid concentration (Y) against the peak area (X). Figure 3 As shown, y = 13.428x - 29.039, R 2 = 0.9968. From Figure 2 The peak area (X) of Lactobacillus gasseri was obtained, and the concentration of indole-3-lactic acid was calculated to be 90.60 μg / mL based on the standard curve.

[0040] Specific Example 3: Analysis of the lactic acid production capacity of Lactobacillus gasseri QS766.

[0041] 1. Preparation of Lactobacillus gasseri seed culture

[0042] Lactobacillus gasseri QS766, stored at -80℃, was taken out and inoculated into MRS liquid medium supplemented with 0.5wt% L-cysteine ​​hydrochloride at a volume ratio of 2% for 3 generations of activation culture.

[0043] 2. Determination of lactic acid, a metabolite of Lactobacillus gasseri

[0044] (1) Seed culture at a volume ratio of 2% was inoculated into 10 mL of MRS medium supplemented with 0.5 wt% L-cysteine ​​hydrochloride and cultured anaerobically at 37℃ for 48 h, repeated 3 times. The culture medium was centrifuged at 4℃ and 8000 rpm / min to collect the supernatant, which was then diluted 1000 times to obtain the sample to be tested. Ion chromatography was used for detection. The chromatographic column was AS11-HC, the mobile phase was KOH, the injection volume was 25 μL, and the flow rate was 1.2 mL / min. The gradient elution program was as follows:

[0045] Table 2 Gradient elution program

[0046]

[0047] (2) The peak area of ​​lactic acid in different concentrations of standard was determined using the external standard method. A standard curve was obtained by plotting the lactic acid concentration (Y) and peak area (X), as follows: Figure 4 As shown, y = 0.162381x + 0.002439, R² = 0.999. Substituting the values ​​from the standard curve, the lactic acid concentration was calculated to be 9.86 g / L.

[0048] Specific Example 4: Detection of the antibacterial activity of Lactobacillus gasseri QS766 against Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, and Gardnerella vaginalis ATCC 14018.

[0049] 1. Preparation of pathogenic bacteria suspension

[0050] Escherichia coli and Staphylococcus aureus were inoculated into LB liquid medium at a volume ratio of 2%, while Gardnerella vaginalis was inoculated into BHI liquid medium containing 5% serum. The cultures were incubated at 37°C for 18-24 hours. The bacterial suspensions, after two generations of activation, were adjusted to an OD concentration using the appropriate liquid medium. 600 It ranges from 0.5 to 0.65.

[0051] 2. Preparation of detection plates

[0052] The adjusted concentrations of *Escherichia coli* and *Staphylococcus aureus* were diluted 1:10 with LB solid medium cooled to approximately 55°C, while *Gardnerella vaginalis* was diluted with BHI solid medium containing 5% serum cooled to approximately 55°C. After thoroughly mixing the pathogenic bacteria with the solid medium, 15 mL was transferred to a sterile petri dish containing an Oxford cup, ensuring the bacterial suspension was evenly spread. Once solidified, the Oxford cup was removed to prepare the test plate.

[0053] 3. Preparation of fermentation supernatant and bacterial suspension of Lactobacillus gasseri QS766

[0054] Lactobacillus gasseri QS766, stored at -80℃, was inoculated at a volume ratio of 2% into MRS liquid medium supplemented with 0.5% L-cysteine ​​hydrochloride and cultured for 3 generations. The completed third-generation Lactobacillus gasseri QS766 culture was centrifuged at 8000 rpm / min for 10 min at 4℃. The supernatant was collected and filtered through a 0.22 μm filter membrane to prepare the fermentation supernatant. The centrifuged bacterial cells were collected, washed 2-3 times with PBS solution, and resuspended in 1 mL of PBS solution to prepare a bacterial suspension.

[0055] 4. Antibacterial activity test

[0056] 200 μL of the prepared *Lactobacillus gasseri* QS766 fermentation supernatant, bacterial suspension, and third-generation culture of *Lactobacillus gasseri* QS766 were added to the wells of Oxford cups on a test plate, and the plates were then incubated at 37°C for 12–18 h. The antibacterial performance of *Lactobacillus gasseri* QS766 was evaluated by measuring the diameter of the inhibition zone. MRS liquid medium containing 0.5% L-cysteine ​​hydrochloride was used as a negative control.

[0057] 5. Evaluation of the antibacterial properties of Lactobacillus gasseri QS766

[0058] The results are as follows Figure 5As shown, the inhibition zone diameters of *Lactobacillus gasseri* QS766 bacterial culture, fermentation supernatant, and bacterial suspension against *Staphylococcus aureus* were 19.5, 14.5, and 15.5 mm, respectively; against *Escherichia coli*, the inhibition zone diameters were 20, 20.5, and 15 mm, respectively; and against *Gardnerella vaginalis*, the inhibition zone diameters were 19.5, 16.5, and 20.5 mm, respectively. This demonstrates that *Lactobacillus gasseri* QS766 exhibits good antibacterial activity in different forms.

[0059] Specific Example 5: In vitro antioxidant evaluation of Lactobacillus gasseri QS766.

[0060] Lactobacillus gasseri QS766 stored at -80℃ was taken out and inoculated into MRS liquid medium supplemented with 0.5% L-cysteine ​​hydrochloride at a volume ratio of 2%. After activation culture for 3 generations, the cells were centrifuged at 8000 rpm / min for 10 min at 4℃. The centrifuged cells were collected and their in vitro antioxidant capacity was tested.

[0061] 1. Evaluation of the superoxide anion scavenging capacity of Lactobacillus gasseri QS766

[0062] The superoxide anion scavenging capacity of Lactobacillus gasseri QS766 was determined according to the instructions of the superoxide anion scavenging kit (GraceBio, Suzhou), and the result was 55.25%. This indicates that Lactobacillus gasseri QS766 has good superoxide anion scavenging capacity.

[0063] 2. Evaluation of the ABTS free radical scavenging ability of Lactobacillus gasseri QS766

[0064] The ABTS free radical scavenging ability of Lactobacillus gasseri QS766 was determined according to the instructions of the ABTS free radical scavenging kit (Grace Biotech, Suzhou), and the result was 40.73%. This indicates that Lactobacillus gasseri QS766 has good ABTS free radical scavenging ability.

[0065] 3. Evaluation of the DPPH free radical scavenging ability of Lactobacillus gasseri QS766

[0066] The DPPH free radical scavenging ability of Lactobacillus gasseri QS766 was determined according to the instructions of the DPPH free radical scavenging ability kit (Grace Biotech, Suzhou), and the result was 29.12%. Therefore, Lactobacillus gasseri QS766 has good DPPH free radical scavenging ability.

[0067] Specific Example 6: Antibiotic susceptibility test of Lactobacillus gasseri QS766.

[0068] 1. Determination of antimicrobial resistance

[0069] After activating and culturing *Lactobacillus gasseri* QS766 for three generations, the bacterial suspension concentration was adjusted with sterile physiological saline. The OD value of the bacterial suspension was measured at 625 nm using a spectrophotometer, ensuring the OD value ranged from 0.16 to 0.20. Following the national food safety standard for the safety evaluation of microbial strains for food use, the antimicrobial resistance of *Lactobacillus gasseri* QS766 was determined using the micro-broth dilution method. Ten types of antimicrobial drugs were tested: tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, vancomycin, penicillin, imipenem, piperacillin, and doxycycline.

[0070] 2. Determination of Antimicrobial Resistance Results

[0071] Based on the national food safety standard for the safety evaluation procedure of microbial strains for food use, the drug resistance of Lactobacillus gasseri QS766 was evaluated. The results are shown in Table 3. Lactobacillus gasseri QS766 showed sensitivity to all of the following types of antimicrobial drugs, indicating that this strain does not have a significant risk of drug resistance and has good biosafety.

[0072] Table 3. Determination of Antimicrobial Resistance of Lactobacillus gasseri QS766

[0073]

[0074] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A strain of *Lactobacillus gasseri* that efficiently utilizes tryptophan to produce indole-3-lactic acid, characterized in that, The strain described is classified and named Lactobacillus gasseri (Lactobacillus gasseri) Lactobacillus gasseri The strain QS766, with accession number CGMCC No.37546, was deposited on January 26, 2026. The depository is the China General Microbiological Culture Collection Center, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

2. The application of *Lactobacillus gasseri* as described in claim 1 in the production of indole-3-lactic acid, characterized in that, The Lactobacillus gasseri strain described herein metabolizes into indole-3-lactic acid after anaerobic fermentation in a tryptophan-containing medium.

3. The application according to claim 2, characterized in that, The tryptophan-containing culture medium is an MRS liquid culture medium containing a final concentration of 1-3 g / L tryptophan and 0.1-1 wt% L-cysteine ​​hydrochloride.

4. The use of Lactobacillus gasseri as described in claim 1 in the preparation of a product for relieving constipation.

5. The use of Lactobacillus gasseri as described in claim 1 in the preparation of products for regulating mood.

6. The use of Lactobacillus gasseri as described in claim 1 in the preparation of products that improve the microecological balance of the female reproductive tract.

7. A microbial preparation comprising Lactobacillus gasseri as described in claim 1, characterized in that, The microbial preparation is selected from Lactobacillus gasseri suspension, lyophilized bacterial powder, or a combination thereof.

8. The use of Lactobacillus gasseri as described in claim 1 or the microbial preparation as described in claim 7 in the preparation of pathogenic bacteria inhibitors, characterized in that, The pathogenic bacteria are selected from at least one of Staphylococcus aureus, Escherichia coli, and Gardnerella vaginalis.

9. The use of Lactobacillus gasseri as described in claim 1 or the microbial preparation as described in claim 7 in the preparation of antioxidants, wherein the antioxidant is a superoxide anion scavenger, an ABTS free radical scavenger, and / or a DPPH free radical scavenger.

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