Lactobacillus rhamnosus with high yield of indole-3-lactic acid and application thereof

Abstract

The application discloses a lactobacillus rhamnosus strain with high indole-3-lactic acid yield and application thereof, and has the characteristics that the strain is named as lactobacillus rhamnosus QS553 strain, the preservation number is CGMCC No.39259, the strain metabolically produces indole-3-lactic acid after anaerobic fermentation in a culture medium containing tryptophan, and the application of the lactobacillus rhamnosus in preparation of products for relieving constipation, anti-tumor products, regulating emotional state products and intestinal micro-ecological balance products is provided, and the application of the strain in preparation of pathogenic bacteria inhibitors and antioxidants is further provided; the advantages are that the strain can efficiently utilize tryptophan to metabolically generate indole-3-lactic acid, has inhibiting effects on escherichia coli, staphylococcus aureus and porphyromonas gingivalis, can eliminate hydroxyl radicals, DPPH free radicals and ABTS free radicals, and has good antioxidant performance.

Description

Technical Field

[0001] This invention relates to Lactobacillus rhamnosus, and more particularly to a strain of Lactobacillus rhamnosus that produces high levels of indole-3-lactic acid and its applications. Background Technology

[0002] Indole-3-lactic acid (ILA) is a key metabolite produced by lactic acid bacteria and bifidobacteria through tryptophan metabolism, exhibiting diverse biological activities in the gut and central nervous system. ILA can regulate intestinal immune homeostasis and barrier function by activating the aryl hydrocarbon receptor (AhR) signaling pathway, reducing peripheral inflammation levels. It also participates in the gut-brain axis regulation at multiple levels by influencing tryptophan metabolic shunting, inhibiting excessive activation of the kynurenine pathway, and regulating vagal nerve signaling and microglial cell inflammatory responses, thus potentially improving depression and anxiety-related behavioral abnormalities. It can also modulate the host's mucosal immune response. Its effects include enhancing epithelial barrier function and inhibiting excessive inflammatory responses by regulating the expression of inflammatory factors (such as IL-10 and TNF-α), maintaining intestinal immune homeostasis, and thus relieving constipation. Studies have shown that ILA exerts anti-tumor effects by targeting RORγt to reduce Th17 cell differentiation, inhibiting Th17 responses, downregulating IL-17 signaling, and improving barrier integrity by activating intestinal epithelial AhR.

[0003] Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus This strain can colonize the intestine and inhibit the adhesion of pathogenic bacteria to the intestinal epithelium through a competitive exclusion mechanism. Fermentation products such as lactic acid from this strain can lower the pH of the intestinal lumen, thereby inhibiting the growth of harmful bacteria and promoting the proliferation of beneficial bacteria, thus maintaining intestinal flora balance and reducing the risk of inflammation and diarrhea. Currently, there are no research reports on *Lactobacillus rhamnosus* that utilize 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 strain of Lactobacillus rhamnosus that produces high levels of indole-3-lactic acid and its applications.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problem is: a strain of *Lactobacillus rhamnosus* that produces indole-3-lactic acid using tryptophan, wherein the *Lactobacillus rhamnosus* is classified as *Lactobacillus rhamnosus* (…). Lacticaseibacillus rhamnosus The strain QS553, with accession number CGMCC No.39259, was deposited on June 16, 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 rhamnosus in the production of indole-3-lactic acid, wherein the Lactobacillus rhamnosus 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 a microbial preparation containing the above-mentioned Lactobacillus rhamnosus, wherein the microbial preparation is selected from bacterial suspension, fermentation supernatant, lyophilized bacterial powder or a combination thereof.

[0009] This invention also provides the use of the above-mentioned *Lactobacillus rhamnosus* or the above-mentioned 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 Porphyromonas gingivalis ( Porphyromonas gingivalis At least one of the following.

[0010] The present invention also provides the use of the above-mentioned Lactobacillus rhamnosus or the above-mentioned microbial preparation in the preparation of antioxidants.

[0011] Furthermore, the antioxidants include hydroxyl radical scavengers, ABTS radical scavengers, and / or DPPH radical scavengers.

[0012] Compared with existing technologies, the advantages of this invention are as follows: This invention discloses a high-yield indole-3-lactic acid (ILA)-producing *Lactobacillus rhamnosus* strain and its applications. This strain can efficiently utilize tryptophan to metabolize and produce indole-3-lactic acid (ILA). Using MRS medium containing 2 g / L tryptophan and 0.5% wt L-cysteine ​​hydrochloride for fermentation, the ILA content can reach 112.10 mg / L. Furthermore, this strain exhibits inhibitory effects on pathogenic bacteria such as *Escherichia coli*, *Staphylococcus aureus*, and *Porphyromonas gingivalis*. It can scavenge hydroxyl radicals, DPPH radicals, and ABTS radicals, demonstrating good antioxidant properties. Under conventional culture conditions, it can produce up to 16.07 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 eight common clinical and food-related antibiotics, including tetracycline, erythromycin, clindamycin, and penicillin, indicating no acquired resistance and high biosafety, making it suitable for the development of food and health-related products.

[0013] In summary, the present invention provides a strain of Lactobacillus rhamnosus QS553 that produces high levels of indole-3-lactic acid, which 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

[0014] Figure 1 Colony morphology of Lactobacillus rhamnosus QS553; Figure 2 Figure showing the results of the ability of Lactobacillus rhamnosus QS553 to produce indole-3-lactic acid; Figure 3 The standard curve for indole-3-lactic acid; Figure 4 This is the standard curve for lactate. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0016] Specific Implementation Example 1: Isolation and Identification of Strains.

[0017] 1. Strains Isolation 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 hours. 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 hours 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 hours. 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 QS553 showed significant advantages in growth performance and acid tolerance. Therefore, strain QS553 was identified as the key strain for this experiment.

[0018] 2. Strain identification 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).

[0019] 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.

[0020] Figure 1 As shown. Based on morphological and 16S rRNA identification, strain QS553 was identified as *Lactobacillus rhamnosus*.

[0021] This strain is currently deposited at the China General Microbiological Culture Collection Center and is classified as *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus The strain QS553, with accession number CGMCCNo.39259, was deposited on June 16, 2026, at 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.

[0022] Specific Example 2: Analysis of the ability of Lactobacillus rhamnosus QS553 to produce indole-3-lactic acid.

[0023] 1. Preparation of Lactobacillus rhamnosus seed culture The Lactobacillus rhamnosus QS553 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.

[0024] 2. Determination of indole-3-lactic acid, a tryptophan metabolite from Lactobacillus rhamnosus (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.

[0025] (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 4.6×150mm 3.5-Micron HPLC system; a detection wavelength of 280nm; mobile phases A (0.1% formic acid aqueous solution) and B (100% acetonitrile); an injection volume of 10μL; and a flow rate of 1mL / min. The gradient elution program was as follows: Table 1 Gradient elution program

[0026] 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 (x) against the peak area (y). Figure 3 As shown, y = 13.428x - 29.039, R 2 = 0.9968. From Figure 2 The peak area (y) of Lactobacillus rhamnosus was obtained, and the concentration of indole-3-lactic acid in the fermentation supernatant of Lactobacillus rhamnosus QS553 was calculated to be 112.10 mg / L based on the standard curve.

[0027] Specific Example 3: Analysis of the lactic acid production capacity of Lactobacillus rhamnosus QS553.

[0028] 1. Preparation of Lactobacillus rhamnosus seed culture The Lactobacillus rhamnosus QS553 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.

[0029] 2. Determination of lactic acid, a metabolite of Lactobacillus rhamnosus (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: Table 2 Gradient elution program

[0030] (2) The peak area of ​​lactic acid in standard samples of different concentrations was determined using the external standard method. A standard curve was obtained by plotting the lactic acid concentration (x) and peak area (y), as follows: Figure 4 As shown, y = 0.1131x - 0.0021, R² = 0.9999. Substituting the values ​​from the standard curve, the concentration of lactic acid in the fermentation supernatant of *Lactobacillus rhamnosus* QS553 was calculated to be 16.07 g / L.

[0031] Specific Example 4: Detection of the antibacterial activity of Lactobacillus rhamnosus QS553 against Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, and Porphyromonas gingivalis ATCC 33277.

[0032] 1. Preparation of pathogenic bacteria suspension Escherichia coli and Staphylococcus aureus were inoculated into LB liquid medium at a volume ratio of 2%, while Porphyromonas gingivalis was inoculated into BHI liquid medium containing 5 vt% serum and incubated at 37°C for 18-24 hours. The bacterial cultures of the activated bacteria from two generations were then adjusted to an OD concentration using the appropriate liquid medium. 600 It ranges from 0.5 to 0.65.

[0033] 2. Preparation of detection plates The adjusted concentrations of *Escherichia coli* and *Staphylococcus aureus* were diluted 1:10 with LB solid medium cooled to approximately 55°C, while *Porphyromonas gingivalis* was diluted with BHI solid medium containing 5 VT% 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.

[0034] 3. Preparation of fermentation supernatant and bacterial suspension of Lactobacillus rhamnosus QS553 Lactobacillus rhamnosus QS553, stored at -80℃, was inoculated at a volume ratio of 2% into MRS liquid medium supplemented with 0.5wt% L-cysteine ​​hydrochloride and cultured for 3 generations. The completed third-generation Lactobacillus rhamnosus QS553 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.

[0035] 4. Antibacterial activity test 200 μL of the prepared *Lactobacillus rhamnosus* QS553 fermentation supernatant, bacterial suspension, and 3-generation culture of *Lactobacillus rhamnosus* QS553 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 rhamnosus* QS553 was evaluated by measuring the diameter of the inhibition zone. MRS liquid medium containing 0.5 wt% L-cysteine ​​hydrochloride was used as a negative control.

[0036] 5. Evaluation of the antibacterial properties of Lactobacillus rhamnosus QS553 The results showed that the inhibition zone diameters of *Lactobacillus rhamnosus* QS553 bacterial culture, fermentation supernatant, and bacterial suspension against *Staphylococcus aureus* were 19.0, 14.5, and 15.0 mm, respectively; against *Escherichia coli*, the inhibition zone diameters were 18.5, 15.0, and 0 mm, respectively; and against *Porphyromonas gingivalis*, the inhibition zone diameters were 18.0, 15.0, and 13.0 mm, respectively. This demonstrates that *Lactobacillus rhamnosus* QS553 exhibits good antibacterial activity in different forms.

[0037] Specific Example 5: In vitro antioxidant evaluation of Lactobacillus rhamnosus QS553.

[0038] Lactobacillus rhamnosus QS553 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%. After activation culture for 3 generations, the cells were centrifuged at 8000rpm / min for 10min at 4℃. The centrifuged cells were collected and their in vitro antioxidant capacity was tested.

[0039] 1. Evaluation of the hydroxyl radical scavenging ability of Lactobacillus rhamnosus QS553 The hydroxyl radical scavenging ability of *Lactobacillus rhamnosus* QS553 was determined according to the instructions of the hydroxyl radical scavenging kit (Grace Biotech, Suzhou), and the result was 48.25%. This indicates that *Lactobacillus rhamnosus* QS553 has good hydroxyl radical scavenging ability.

[0040] 2. Evaluation of the ABTS free radical scavenging ability of Lactobacillus rhamnosus QS553 The ABTS free radical scavenging ability of *Lactobacillus rhamnosus* QS553 was determined according to the instructions of the ABTS free radical scavenging kit (Grace Biotech, Suzhou), and the result was 41.41%. This indicates that *Lactobacillus rhamnosus* QS553 has good ABTS free radical scavenging ability.

[0041] 3. Evaluation of the DPPH free radical scavenging ability of Lactobacillus rhamnosus QS553 Following the instructions of the DPPH free radical scavenging ability kit (Grace Biotech, Suzhou), the DPPH free radical scavenging ability of Lactobacillus rhamnosus QS553 was determined, and the result was 91%. Therefore, Lactobacillus rhamnosus QS553 exhibits good DPPH free radical scavenging ability.

[0042] Specific Example 6: Antibiotic Sensitivity Test of Lactobacillus rhamnosus QS553.

[0043] 1. Determination of antimicrobial resistance After activating and culturing *Lactobacillus rhamnosus* QS553 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 rhamnosus* QS553 was determined using the micro-broth dilution method. The following 11 antimicrobial types were tested: gentamicin, kanamycin, tetracycline, erythromycin, chloramphenicol, ampicillin, vancomycin, penicillin, imipenem, piperacillin, and doxycycline.

[0044] 2. Determination of Antimicrobial Resistance Results Based on the national food safety standard for the safety evaluation procedure of microbial strains, the drug resistance of Lactobacillus rhamnosus QS553 was evaluated. The results are shown in Table 3. Lactobacillus rhamnosus QS553 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.

[0045] Table 3. Determination of Antimicrobial Resistance of Lactobacillus rhamnosus QS553

[0046] 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 rhamnosus* that produces high levels of indole-3-lactic acid, characterized in that, The Lactobacillus rhamnosus mentioned above is classified as Lactobacillus rhamnosus (Lactobacillus rhamnosus). Lacticaseibacillus rhamnosus The strain is QS553, with the preservation number CGMCC No.39259.

2. The application of *Lactobacillus rhamnosus* as described in claim 1 in the production of indole-3-lactic acid, characterized in that, The *Lactobacillus rhamnosus* strain 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. A microbial preparation comprising *Lactobacillus rhamnosus* as described in claim 1, characterized in that, The microbial preparation is selected from Lactobacillus rhamnosus bacterial suspension, fermentation supernatant, freeze-dried bacterial powder, or a combination thereof.

5. The use of *Lactobacillus rhamnosus* according to claim 1 or the microbial preparation according to claim 4 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 Porphyromonas gingivalis.

6. The use of *Lactobacillus rhamnosus* according to claim 1 or the microbial preparation according to claim 4 in the preparation of antioxidants, characterized in that... The antioxidants mentioned are hydroxyl radical scavengers, ABTS radical scavengers, and / or DPPH radical scavengers.

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