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

Abstract

The application discloses a lactobacillus helveticus strain with high indole-3-lactic acid yield and application thereof, and has the characteristics that the strain is named as the lactobacillus helveticus QS666 strain, the preservation number is CGMCC No. 39401, the strain metabolically produces indole-3-lactic acid after anaerobic fermentation in a culture medium containing tryptophan, and the application of the lactobacillus helveticus in preparing a constipation relieving product, an anti-tumor product, a mood state regulating product and an intestinal micro-ecological balance product is provided, and the application of the strain in preparing a pathogenic bacterium inhibitor and an antioxidant 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 helveticus, and more particularly to a strain of Lactobacillus helveticus 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 using tryptophan metabolism, exhibiting diverse biological activities in the gut and central nervous system. ILA acts as a crosstalk mechanism between intestinal epithelial cells and macrophages to maintain intestinal homeostasis, alleviate intestinal barrier damage, reduce inflammatory responses in intestinal epithelial cells, and relieve constipation. Recent studies have shown that ILA can guide the differentiation of immature colonic macrophages, thereby reducing the incidence of colitis-related tumors. Furthermore, ILA has been shown to enhance CD8 T cell function through epigenetic mechanisms, thereby inhibiting tumor growth. ILA can cross the blood-brain barrier to enter the brain, activating the aryl hydrocarbon receptor (AHR) pathway, thereby suppressing neuroinflammation and alleviating depressive symptoms.

[0003] Lactobacillus helveticus ( Lactobacillus helveticus This strain can temporarily colonize the intestine by adhering to the mucins and glycosides on the surface of intestinal epithelial cells, thereby gaining a competitive advantage in the intestinal lumen and inhibiting the adhesion and colonization of pathogens such as Escherichia coli and Salmonella. Simultaneously, this strain can promote the expression of intestinal epithelial tight junction proteins (such as Occludin, Claudin-1, and ZO-1), enhancing intestinal barrier function and reducing intestinal permeability, thus maintaining intestinal homeostasis. It also exhibits significant antibacterial activity by secreting lactic acid, hydrogen peroxide, and antimicrobial peptides (such as Helveticin), inhibiting the growth of various harmful bacteria and thus alleviating intestinal inflammatory responses. Currently, there are no research reports on Lactobacillus helveticus 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 strain of Lactobacillus helveticus 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 problems is: a strain of *Lactobacillus helveticus* that utilizes tryptophan to produce high levels of indole-3-lactic acid, and the strain is classified and named *Lactobacillus helveticus* (…). Lactobacillus helveticus The strain QS666, with accession number CGMCC No.39401, 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 helveticus in the production of indole-3-lactic acid, wherein the Lactobacillus helveticus 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 helveticus in the preparation of products for relieving constipation.

[0009] The present invention also provides the application of the above-mentioned Lactobacillus helveticus in the preparation of antitumor products.

[0010] The present invention also provides the application of the above-mentioned Lactobacillus helveticus in the preparation of intestinal flora regulator products.

[0011] The present invention also provides the application of the above-mentioned Lactobacillus helveticus in the preparation of products for regulating mood.

[0012] The present invention also provides a microbial preparation containing the above-mentioned Lactobacillus helveticus, wherein the microbial preparation is selected from bacterial suspension, fermentation supernatant, lyophilized bacterial powder or a combination thereof.

[0013] This invention also provides the use of the above-mentioned Lactobacillus helveticus or the above-mentioned microbial preparation in the preparation of a pathogenic bacteria inhibitor, 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.

[0014] The present invention also provides the use of the above-mentioned Lactobacillus helveticus or the above-mentioned microbial preparation in the preparation of antioxidants, wherein the antioxidants include hydroxyl radical scavengers, ABTS radical scavengers and / or DPPH radical scavengers.

[0015] Compared with existing technologies, the advantages of this invention are as follows: This invention discloses a strain of *Lactobacillus helveticus* that utilizes tryptophan to produce high levels of 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 wt% L-cysteine ​​hydrochloride for fermentation, the ILA content can reach 266.49 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 15.89 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 11 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 present invention provides a strain of Lactobacillus helveticus QS666 that produces indole-3-lactic acid. This strain 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, antibacterial agents, and antioxidant product development. Attached Figure Description

[0017] Figure 1 Image of Lactobacillus helveticus QS666 colony morphology; Figure 2 Liquid chromatography peaks for indole-3-lactic acid production by Lactobacillus helveticus QS666; Figure 3 The standard curve for indole-3-lactic acid; Figure 4 This is the standard curve for lactate. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0019] Specific Implementation Example 1: Isolation and Identification of Strains.

[0020] 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 -6Three 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 QS666 showed significant advantages in growth performance and acid tolerance. Therefore, strain QS666 was identified as the key strain for this experiment.

[0021] 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).

[0022] 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.

[0023] Figure 1 As shown. Based on morphological and 16S rRNA identification, strain QS666 was identified as Lactobacillus helveticus.

[0024] This strain is currently deposited at the China General Microbiological Culture Collection Center and is classified as *Lactobacillus helveticus*. Lactobacillus helveticus The strain QS666, with accession number CGMCC No.39401, was deposited on June 16, 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.

[0025] Specific Example 2: Analysis of the ability of Lactobacillus helveticus QS666 to produce indole-3-lactic acid.

[0026] 1. Preparation of Lactobacillus helveticus seed culture Lactobacillus helveticus QS666 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.

[0027] 2. Determination of indole-3-lactic acid, a tryptophan metabolite from Lactobacillus helveticus (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.

[0028] (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

[0029] 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 helveticus was obtained, and the concentration of indole-3-lactic acid in the fermentation supernatant of Lactobacillus helveticus QS666 was calculated to be 266.49 mg / L based on the standard curve.

[0030] Specific Example 3: Analysis of the lactic acid production capacity of Lactobacillus helveticus QS666.

[0031] 1. Preparation of Lactobacillus helveticus seed culture Lactobacillus helveticus QS666 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.

[0032] 2. Determination of lactic acid, a metabolite of Lactobacillus helveticus (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

[0033] (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 lactic acid concentration was calculated to be 15.89 g / L.

[0034] Specific Example 4: Detection of the antibacterial activity of Lactobacillus helveticus QS666 against Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, and Porphyromonas gingivalis ATCC 33277.

[0035] 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 at a volume ratio of 2%. 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 corresponding liquid medium. 600 It ranges from 0.5 to 0.65.

[0036] 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 1:10 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. After solidification, the Oxford cup was removed to prepare the test plate.

[0037] 3. Preparation of fermentation supernatant and bacterial suspension of Lactobacillus helveticus QS666 Lactobacillus helveticus QS666, 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 helveticus QS666 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.

[0038] 4. Antibacterial activity test 200 μL of the prepared *Lactobacillus helveticus* QS666 fermentation supernatant, bacterial suspension, and third-generation culture of *Lactobacillus helveticus* QS666 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 helveticus* QS666 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.

[0039] 5. Evaluation of the antibacterial properties of Lactobacillus helveticus QS666 The results showed that the inhibition zone diameters of *Lactobacillus helveticus* QS666 bacterial culture, fermentation supernatant, and bacterial suspension against *Staphylococcus aureus* were 18, 14.5, and 13.5 mm, respectively; against *Escherichia coli*, the inhibition zone diameters were 19.5, 16.5, and 0 mm, respectively; and against *Porphyromonas gingivalis*, the inhibition zone diameters were 16, 13, and 13.5 mm, respectively. This demonstrates that *Lactobacillus helveticus* QS666 exhibits good antibacterial activity in different forms.

[0040] Specific Example 5: In vitro antioxidant evaluation of Lactobacillus helveticus QS666.

[0041] Lactobacillus helveticus QS666 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 and 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.

[0042] 1. Evaluation of the hydroxyl radical scavenging ability of Lactobacillus helveticus QS666 The hydroxyl radical scavenging ability of Lactobacillus helveticus QS666 was determined according to the instructions of the hydroxyl radical scavenging kit (Grace Biotech, Suzhou), and the result was 90%. This indicates that Lactobacillus helveticus QS666 has good hydroxyl radical scavenging ability.

[0043] 2. Evaluation of the ABTS free radical scavenging ability of Lactobacillus helveticus QS666 The ABTS free radical scavenging ability of Lactobacillus helveticus QS666 was determined according to the instructions of the ABTS free radical scavenging kit (Grace Biotech, Suzhou), and the result was 40%. This indicates that Lactobacillus helveticus QS666 has good ABTS free radical scavenging ability.

[0044] 3. Evaluation of the DPPH free radical scavenging ability of Lactobacillus helveticus QS666 Following the instructions of the DPPH free radical scavenging ability kit (Grace Biotech, Suzhou), the DPPH free radical scavenging ability of Lactobacillus helveticus QS666 was determined, and the result was 35%. Therefore, Lactobacillus helveticus QS666 exhibits good DPPH free radical scavenging ability.

[0045] Specific Example 6: Antibiotic Sensitivity Test of Lactobacillus helveticus QS666.

[0046] 1. Determination of antimicrobial resistance After activating and culturing *Lactobacillus helveticus* QS666 for three generations, the bacterial 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 helveticus* QS666 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.

[0047] 2. Determination of Antimicrobial Resistance Results The drug resistance of Lactobacillus helveticus QS666 was evaluated according to the National Food Safety Standard for Food-grade Microbial Strains. The results are shown in Table 3. Lactobacillus helveticus QS666 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.

[0048] Table 3. Determination of Antimicrobial Resistance of Lactobacillus helveticus QS666

[0049] 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 type of Lactobacillus helveticus that produces high levels of indole-3-lactic acid, characterized in that, The strain described is classified and named Lactobacillus helveticus ( Lactobacillus helveticus The strain QS666, with preservation number CGMCC No.39401, is a 100-year-old, 200-year-old strain.

2. The application of *Lactobacillus helveticus* as described in claim 1 in the production of indole-3-lactic acid, characterized in that, The aforementioned Lactobacillus helveticus strain metabolizes to produce 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 the Lactobacillus helveticus according to claim 1 in the preparation of a product for relieving constipation.

5. The use of Lactobacillus helveticus as described in claim 1 in the preparation of antitumor products.

6. The use of Lactobacillus helveticus as described in claim 1 in the preparation of intestinal flora regulator products.

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

8. A microbial preparation comprising *Lactobacillus helveticus* as described in claim 1, characterized in that, The microbial preparation is selected from Lactobacillus helveticus suspension, fermentation supernatant, freeze-dried bacterial powder, or a combination thereof.

9. The use of the *Lactobacillus helveticus* of claim 1 or the microbial preparation of claim 8 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.

10. The use of the *Lactobacillus helveticus* of claim 1 or the microbial preparation of claim 8 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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