Lactobacillus helveticus hp-b1137 and application thereof

Abstract

The application discloses a lactobacillus helveticus HP-B1137 and application thereof, and belongs to the technical field of biotechnology.The lactobacillus helveticus HP-B1137 is preserved in the China Center for Type Culture Collection on July 30, 2026, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20261780.The lactobacillus helveticus HP-B1137 provided by the application has good gastric acid tolerance, high-temperature tolerance, bile salt tolerance, good acid production capacity and physiological and biochemical capacity suitable for the gastrointestinal tract of an organism, and has good bacteriostatic effects on bacillus cereus, klebsiella pneumoniae, acinetobacter baumannii, proteus mirabilis, drug-resistant acinetobacter baumannii, drug-resistant escherichia coli, salmonella typhi, escherichia coli, drug-resistant pseudomonas aeruginosa, drug-resistant staphylococcus aureus and shigella.Meanwhile, the lactobacillus helveticus HP-B1137 has the ability to convert calycosin-7-glucoside into calycosin.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of Lactobacillus helveticus HP-B1137 and its applications. Background Technology

[0002] Lactobacillus helveticus is a type of Gram-positive bacterium that is a probiotic. It is rod-shaped, non-flagellated, non-spore-forming, with round, milky-white colonies and regular edges. It is chemoheterotrophic, facultatively anaerobic, and does not liquefy gelatin.

[0003] Fermented milk containing *Lactobacillus helveticus* contains various bioactive peptides that enhance metabolism, boost immunity, act as enzyme inhibitors, and possess antibacterial, antiviral, and lipid-lowering properties. The proteases secreted by *Lactobacillus helveticus* release the bioactive peptides from the porin in dairy products, effectively lowering blood pressure, relieving tension, improving brain function, and enhancing memory and learning abilities. Furthermore, fermented milk containing *Lactobacillus helveticus* also contains casein phosphopeptides (CPPs), which promote the absorption of elements such as calcium and iron.

[0004] Astragalus is a commonly used traditional Chinese medicine with effects such as tonifying qi and raising yang, consolidating the exterior and stopping sweating, and promoting diuresis and reducing edema. Its main active components are saponins and flavonoids. Verbascoside and verbascoside are the two main compounds in Astragalus. Verbascoside is a natural flavonoid compound with various biological activities, including antioxidant, anti-inflammatory, antibacterial, and antitumor effects, and can be obtained through extraction and purification of natural plant materials. Verbascoside is a natural compound with antioxidant and anti-inflammatory activities, which can enhance immune function, antioxidant, anti-radiation, and anti-cancer effects. Although both have antioxidant effects, verbascoside has a greater antioxidant capacity than verbascoside. Verbascoside is relatively scarce in Astragalus and is difficult to extract. Therefore, it is crucial to biotransform verbascoside to remove the glycosyl group and obtain verbascoside, thereby improving its antioxidant capacity. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Lactobacillus helveticus HP-B1137 and its applications, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a strain of Lactobacillus helveticus ( Lactobacillus helveticus HP-B1137, the Lactobacillus helveticus HP-B1137, was deposited at the China Center for Type Culture Collection (CCTCC) on July 30, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261780.

[0007] The second technical solution of the present invention is a microbial preparation comprising the aforementioned Lactobacillus helveticus HP-B1137.

[0008] The third technical solution of the present invention is the application of the Lactobacillus helveticus HP-B1137 or the microbial preparation in the preparation of antibacterial products, wherein the antibacterial products are used to inhibit at least one of Bacillus cereus, Klebsiella pneumoniae, Acinetobacter baumannii, Proteus mirabilis, drug-resistant Acinetobacter baumannii, drug-resistant Escherichia coli, Salmonella typhi, Escherichia coli, drug-resistant Pseudomonas aeruginosa, drug-resistant Staphylococcus aureus, and Shigella.

[0009] The fourth technical solution of the present invention is an antibacterial product, comprising the aforementioned Lactobacillus helveticus HP-B1137 or the aforementioned microbial preparation.

[0010] The fifth technical solution of the present invention is the application of Lactobacillus helveticus HP-B1137 or the microbial preparation in the conversion of verbascoside glucoside into verbascoside.

[0011] The sixth technical solution of the present invention is a method for preparing vernix isoflavone, comprising the following steps: mixing the aforementioned Lactobacillus helveticus HP-B1137 or the aforementioned microbial preparation with vernix isoflavone glucoside, performing anaerobic culture, and separating vernix isoflavone from the fermentation product after the culture is completed.

[0012] Based on the above technical solution, the present invention has the following technical effects: The *Lactobacillus helveticus* HP-B1137 provided by this invention exhibits good tolerance to gastric acid, high temperature, and bile salts, as well as good acid production and adaptability to the physiological and biochemical processes of the gastrointestinal tract. It shows good antibacterial effects against *Bacillus cereus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Proteus mirabilis*, drug-resistant *Acinetobacter baumannii*, drug-resistant *Escherichia coli*, *Salmonella typhi*, *Escherichia coli*, drug-resistant *Pseudomonas aeruginosa*, drug-resistant *Staphylococcus aureus*, and *Shigella*. Simultaneously, it possesses the ability to convert verbascoside glucoside into verbascoside. The biotransformation method also offers advantages such as high reaction selectivity, mild conditions, few byproducts (no impurity peaks in the liquid chromatography), simple post-processing (the product contains only substrate and product, simplifying post-processing), and low environmental pollution (biocatalysis has low environmental impact), making it one of the important routes for obtaining verbascoside. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is a diagram showing the growth morphology of Lactobacillus helveticus strain HP-B1137. Figure 2 Agarose gel electrophoresis image of the PCR products obtained by PCR amplification of Lactobacillus helveticus HP-B1137 16S rRNA; M: 2K plus II DNA marker; 1, 2: PCR amplification products of Lactobacillus helveticus HP-B1137 16S rRNA. Figure 3 The gene of Lactobacillus helveticus HP-B1137 16S rRNA gel-recovered product was recombined into Escherichia coli competent cells GB05-dir. After culturing in solid medium for a period of time, monoclonal antibodies were picked and PCR amplified. The agarose gel electrophoresis image of the monoclonal antibody PCR product is shown. M: 2K plus II DNA marker; 1, 2: monoclonal antibody PCR product. Figure 4 Color changes in carbohydrate fermentation experiment of Lactobacillus helveticus HP-B1137; 1-11 respectively used aescin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose and 1% sodium hippurate as carbon sources. Figure 5 The image shows the antibacterial effect of Lactobacillus helveticus HP-B1137 fermentation broth against drug-resistant Acinetobacter baumannii (A), Klebsiella pneumoniae (B), Acinetobacter baumannii (C), drug-resistant Staphylococcus aureus (D), drug-resistant Escherichia coli (E), Salmonella typhi (F), Shigella (G), drug-resistant Pseudomonas aeruginosa (H), Bacillus cereus (I), Escherichia coli (J), and Proteus mirabilis (K). In the image, F in the petri dish represents the uncentrifuged bacterial culture, and S represents the supernatant after centrifugation. Figure 6 The complete genome map of Lactobacillus helveticus HP-B1137; Figure 7 The image shows plasmid 1 of Lactobacillus helveticus HP-B1137. Figure 8 The plasmid 2 map of Lactobacillus helveticus HP-B1137; Figure 9 This is a statistical chart of shared and unique annotations for gene coding databases. The left side of the chart shows the gene annotation statistics for general databases, while the right side shows the shared and unique statistics. For example, the first column shows the shared statistics for all general databases, the top column shows the number of entries, and the bottom column shows the annotations for shared or unique databases. Figure 10 This is a statistical diagram of PFAM domains; the horizontal axis represents the name of the protein family, and the vertical axis represents the number of genes that were mapped to that protein family. Figure 11 A classification diagram of GO annotation results; the horizontal axis represents the content of each GO category, and the vertical axis represents the number of genes. Figure 12 This is a classification diagram of KEGG pathway results; the horizontal axis represents the number of genes annotated under each pathway category; the vertical axis represents the pathway category, with different colors representing different major categories. Figure 13 This is a classification chart of COG results; the horizontal axis represents the COG classification content, and the vertical axis represents the number of genes. Figure 14 This is a distribution map of species in the National Northwest (NR) region; different colors in the map represent different species. Figure 15 This is a classification chart of TCDB results; the horizontal axis represents the TCDB classification content, and the vertical axis represents the number of genes. Figure 16 This is a high-performance liquid chromatography (HPLC) chromatogram of the conversion of verbascoside to verbascoside by Lactobacillus helveticus HP-B1137.

[0015] Figure 17 This is a phylogenetic tree of Lactobacillus helveticus HP-B1137. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0022] This invention investigates and analyzes the antibacterial effects of *Lactobacillus helveticus* HP-B1137 on multiple pathogenic bacteria through observation of bacterial growth status, evaluation of temperature tolerance, acid production capacity, acid resistance, bile salt tolerance, and the effects of its bacterial suspension and fermentation products on these bacteria. The results show that *Lactobacillus helveticus* HP-B1137 exhibits strong growth and reproduction capabilities, high temperature tolerance, strong acid production capacity, strong acid resistance, and strong bile salt tolerance. The bacterial suspension and fermentation products demonstrate a certain degree of resistance to early microbial infections and show good inhibitory effects against *Bacillus cereus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Proteus mirabilis*, drug-resistant *Acinetobacter baumannii*, drug-resistant *Escherichia coli*, *Salmonella typhi*, *Escherichia coli*, drug-resistant *Pseudomonas aeruginosa*, drug-resistant *Staphylococcus aureus*, and *Shigella*. Finally, this invention performs whole-genome sequencing on *Lactobacillus helveticus* HP-B1137, providing a foundation for further in-depth research on this bacteria.

[0023] This invention has revealed that *Lactobacillus helveticus* HP-B1137 possesses excellent growth and reproductive capabilities, good tolerance to high temperatures, superior acid production, excellent resistance to gastric acid, and extremely high tolerance to bile salts. It can relatively adapt to the complex gastrointestinal environment and exhibits certain immunomodulatory effects. Furthermore, it can efficiently convert verbascoside glucoside into verbascoside within a short period, thereby enhancing its antioxidant capacity.

[0024] The bacterial culture of Lactobacillus helveticus HP-B1137 and its fermentation products provided by this invention can convert verbascoside glucoside into verbascoside, which can be used in the production of food and health products using Astragalus membranaceus as a raw material.

[0025] This invention provides a strain of Lactobacillus helveticus ( Lactobacillus helveticus HP-B1137, the Lactobacillus helveticus HP-B1137, was deposited at the China Center for Type Culture Collection (CCTCC) on July 30, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261780.

[0026] This invention also provides a microbial preparation comprising the aforementioned Lactobacillus helveticus HP-B1137.

[0027] The present invention also provides the application of the aforementioned Lactobacillus helveticus HP-B1137 or the aforementioned microbial preparation in the preparation of antibacterial products, wherein the antibacterial products are used to inhibit at least one of Bacillus cereus, Klebsiella pneumoniae, Acinetobacter baumannii, Proteus mirabilis, Salmonella typhi, Escherichia coli, drug-resistant Pseudomonas aeruginosa, drug-resistant Staphylococcus aureus, and Shigella.

[0028] This invention also provides an antibacterial product, comprising the aforementioned Lactobacillus helveticus HP-B1137 or the aforementioned microbial preparation.

[0029] In some specific implementations, the antibacterial product is used to inhibit at least one of Bacillus cereus, Klebsiella pneumoniae, Acinetobacter baumannii, Proteus mirabilis, drug-resistant Acinetobacter baumannii, drug-resistant Escherichia coli, Salmonella typhi, Escherichia coli, drug-resistant Pseudomonas aeruginosa, drug-resistant Staphylococcus aureus, and Shigella.

[0030] This invention also provides the application of the aforementioned Lactobacillus helveticus HP-B1137 or the aforementioned microbial preparation in the conversion of verbascoside glucoside into verbascoside.

[0031] This invention also provides a method for preparing verbascoside, comprising the following steps: mixing the aforementioned Lactobacillus helveticus HP-B1137 or the aforementioned microbial preparation with verbascoside glucoside, performing anaerobic culture, and separating verbascoside from the fermentation product after the culture is completed.

[0032] In some specific implementations, the anaerobic culture method is as follows: add verrucoside isoflavone glucoside to the culture medium of Lactobacillus helveticus HP-B1137 and culture anaerobically at 180 r / min for 24-36 h.

[0033] In some specific implementations, the concentration of the verbascoside added to the culture medium is 0.2 mmol / L.

[0034] Example 1 Isolation, purification and identification of Lactobacillus helveticus HP-B1137: (1) Isolation and purification of Lactobacillus helveticus HP-B1137 This bacterial strain originated from Heze City, Shandong Province. The specific steps for its isolation and purification were as follows: First, the petri dishes, bamboo sticks, triangular rakes, pipette tips, MRS medium, and physiological saline were sterilized. Then, the MRS medium was poured into the sterilized petri dishes to form MRS agar plates. Next, the bacterial seed culture was taken and diluted to 10⁻⁶ with physiological saline. -5 10 -6 10 -7 To determine the concentration, the dilution was transferred to MRS solid plates using a pipette and spread using a triangular rake. After spreading, the plates were placed in a 37°C incubator for anaerobic incubation. After two days of incubation, colony characteristics were observed. Single colonies exhibiting the typical characteristics of being "milky white, round, smooth and moist, with neat edges" were selected and inoculated onto new MRS plates using a bamboo skewer for streaking purification. The incubation and selection process was repeated until a pure culture of the strain was obtained. The purified strain was identified and preliminarily identified as *Lactobacillus helveticus*. The pure culture was preserved for subsequent research and use. The growth morphology of the strain is shown in the figure below. Figure 1 As shown, by Figure 1 It can be seen that the colonies of this strain are round, milky white, with neat edges, and the bacterial cells are typically long rod-shaped, which is consistent with the morphological characteristics of Lactobacillus helveticus.

[0035] (2) Identification of Lactobacillus helveticus HP-B1137 16S rRNA by whole-sequencing The primer sequences used for identification are as follows: 27F-PBACS, SEQ ID NO.1: 5′-GGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCGAGAGTTTGATCCTGGCTCAG-3′; 1541R-PBACS, SEQ ID NO.2: 5′-GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCAAGGAGGTGATCCAGCCGCA-3′; T7-Elong, SEQ ID NO.3: 5′-TTAATACGACTCACTATAGGGGAATTG-3′; T7-TermElong, SEQ ID NO.4: 5′-TTATGCTAGTTATTGCTCAGCGG-3′.

[0036] The purified bacterial strain was selected, and the strain DNA fragment was obtained by SDS-alkali lysis. The 16S rRNA of the strain was amplified by PCR using 16S primers 27F-PBACS and 1541R-PBACS. The PCR amplification conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, for 30 cycles; 72℃ extension for 10 min; and storage at 16℃.

[0037] The obtained PCR products were identified by agarose gel electrophoresis. Figure 2 The gene from the recovered gel was recombined into *E. coli* competent cells GB05-dir. After a period of solid culture, monoclonal antibodies were picked and amplified by PCR using 16S primers T7-Elong and T7-TermElong. The PCR conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, 30 cycles; 72℃ extension for 10 min; and storage at 16℃. The PCR products were identified by agarose gel electrophoresis. Figure 3 The PCR product was sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the strain's 16S rRNA is as follows:

[0038] BLAST gene alignment showed that the HP-B1137 16S rRNA had a 99.62% similarity to Lactobacillus helveticus LH5 in GenBank, indicating that it is a Lactobacillus helveticus strain. Lactobacillus helveticus The strain was named HP-B1137. A phylogenetic tree was constructed to compare HP-B1137 with that of *Lactobacillus helveticus*, as shown below. Figure 17 As shown.

[0039] (3) Carbohydrate fermentation experiment of Lactobacillus helveticus HP-B1137 HBI lactic acid bacteria biochemical identification strips were used to identify the response of *Lactobacillus helveticus* HP-B1137 to 11 carbohydrates. Color changes were observed as follows: Figure 4 As shown in Table 1, the strain showed positive reactions to aescin, cellobiose, maltose, salicin, sucrose, inulin, and lactose, and negative reactions to mannitol, sorbitol, raffinose, and 1% sodium hippurate. Its carbohydrate fermentation characteristics were consistent with the typical characteristics of Lactobacillus helveticus. Combined with colony morphology observation and molecular identification results, it was further confirmed that the strain was Lactobacillus helveticus.

[0040] Table 1. Results of carbohydrate fermentation experiment using Lactobacillus helveticus HP-B1137 Example 2 Evaluation of the growth status of Lactobacillus helveticus HP-B1137: Using a bamboo skewer, a single colony of *Lactobacillus helveticus* HP-B1137 from MRS solid medium was inoculated into freshly sterilized MRS liquid medium and anaerobically cultured at 37°C and 180 rpm for 24 h. One mL of fermentation broth was pipetteted, centrifuged at 8000 rpm for 1 min, the supernatant was discarded, and the bacterial cells were weighed to calculate the wet weight. After drying, the cells were weighed again to calculate the dry weight.

[0041] The dry weight of the bacterial cells obtained by weighing was 4.75 × 10⁻⁶. -3 ±0.00065 g / mL (Table 2), indicating that Lactobacillus helveticus HP-B1137 has a strong reproductive capacity and can reproduce in large quantities.

[0042] Table 2. Study on the growth status of Lactobacillus helveticus HP-B1137 strain. Example 3 Evaluation of the temperature tolerance of Lactobacillus helveticus HP-B1137: Single colonies of *Lactobacillus helveticus* HP-B1137 from MRS solid medium were inoculated onto freshly sterilized MRS liquid medium using bamboo sticks and anaerobically cultured at 37°C and 180 r / min for 20 h. The bacterial solutions were then heated at 37°C, 50°C, and 70°C for 1 min, 2 min, and 3 min, respectively. The heated solutions were then spread onto solid MRS medium and anaerobically cultured at 37°C for 20 h. Single colonies on the plates were then counted.

[0043] A large number of viable bacteria of Lactobacillus helveticus HP-B1137 were still present after heating at 70℃ for 1 min (Table 3), indicating that Lactobacillus helveticus HP-B1137 has good heat resistance.

[0044] Table 3. Temperature tolerance study of Lactobacillus helveticus HP-B1137 Example 4 Evaluation of the acid-producing capacity of Lactobacillus helveticus HP-B1137: Single colonies of *Lactobacillus helveticus* HP-B1137 from MRS solid medium were inoculated onto freshly sterilized MRS liquid medium using bamboo sticks and cultured anaerobically at 37°C and 180 r / min. The pH of the supernatant was measured after centrifugation at 3580 r / min for 15 min at 12 h, 21 h, and 26 h.

[0045] Lactobacillus helveticus HP-B1137 can reach acidity after 12 h of anaerobic culture, indicating that Lactobacillus helveticus HP-B1137 has a strong acid-producing ability (Table 4).

[0046] Table 4. Acid-producing capacity of Lactobacillus helveticus HP-B1137 Example 5 Evaluation of the acid resistance of Lactobacillus helveticus HP-B1137: Prepare artificial gastric solution (8.2 mL dilute hydrochloric acid, 400 mL double-distilled water, 5 g pepsin; shake well, then add double-distilled water to a final volume of 500 mL; adjust pH to 2.7, 3.46, and 4.75 using a measured amount of the solution. Sterilize using a 0.44 μm filter membrane and store at 4°C for later use). Inoculate single colonies of *Lactobacillus helveticus* HP-B1137 from MRS solid medium onto freshly sterilized MRS liquid medium using a bamboo stick, and anaerobically incubate at 37°C and 180 rpm for 20 h. Centrifuge 60 mL of the bacterial suspension at 3580 rpm for 15 min, discard the supernatant, and resuspend the bacteria in 1.5 mL of physiological saline. Add 6 mL of artificial gastric solution to three centrifuge tubes, followed by 200 μL of the resuspended bacterial suspension, and mix well. Spread the mixture onto solid MRS medium at 0 min and 150 min, and incubate for 20 h. Count the single colonies on the plates and calculate the viability.

[0047] Lactobacillus helveticus HP-B1137 still had a large number of surviving strains in artificial gastric fluid at pH 2.7 after 150 min, indicating that it has strong acid resistance (Table 5).

[0048] Table 5. Acid resistance study of Lactobacillus helveticus HP-B1137 Example 6 Evaluation of bile salt tolerance in Lactobacillus helveticus HP-B1137: Prepare artificial intestinal fluid (3.4 g potassium dihydrogen phosphate dissolved in 250 mL double-distilled water, pH adjusted to 6.8 with 0.1 mol / L sodium hydroxide solution, 2.5 g trypsin added, shaken well, and then diluted to 500 mL with double-distilled water. A certain amount of the solution was then mixed with ox bile salts to prepare artificial intestinal fluids with bile salt concentrations of 0.03%, 0.1%, 0.2%, and 0.3%. Sterilize using a 0.44 μm filter membrane and store at 4℃ for later use). Inoculate single colonies of *Lactobacillus helveticus* HP-B1137 from MRS solid medium onto freshly sterilized MRS liquid medium using a bamboo stick. Incubate anaerobicly at 37℃ and 180 rpm for 20 h. Centrifuge 60 mL of the bacterial suspension at 3580 rpm for 15 min, discard the supernatant, and resuspend the bacteria in 1.5 mL of physiological saline. Add 6 mL of artificial intestinal fluid to each of four centrifuge tubes, followed by 200 μL of the resuspended bacterial suspension and mix well. Diluted to 0.2 mmol / L at 0 min and 150 min, the culture was plated onto solid MRS medium and incubated for 20 h. Single colonies on the plates were counted and the survival rate was calculated (Table 6). As shown in Table 6, the survival rate of the strain gradually decreased with increasing bile salt concentration. The survival rate was as high as 99.65% at a bile salt concentration of 0.03%, and still reached 18.87% at 0.3%, indicating that Lactobacillus helveticus HP-B1137 has good tolerance to bile salts.

[0049] Table 6. Study on bile salt tolerance of Lactobacillus helveticus HP-B1137 Example 7 Evaluation of the antibacterial effect of Lactobacillus helveticus HP-B1137 fermentation broth: Using a bamboo stick, inoculate a single colony of *Lactobacillus helveticus* HP-B1137 from MRS solid medium into freshly sterilized MRS liquid medium and anaerobically culture at 37°C and 180 r / min for 20 h. Take 10 mL of the bacterial culture, centrifuge at 8000 r / min for 1 min, and collect the supernatant. Reserve another 1 mL of the bacterial culture.

[0050] Bacillus cereus, Klebsiella pneumoniae, Acinetobacter baumannii, Proteus mirabilis, drug-resistant Acinetobacter baumannii, drug-resistant Escherichia coli, Salmonella typhi, Escherichia coli, drug-resistant Pseudomonas aeruginosa, drug-resistant Staphylococcus aureus, and Shigella were mixed separately on LB agar plates. The mixtures were poured onto LB agar plates and, after the plates had slightly dried, evenly distributed into four 8.0 mm diameter Oxford cups. The plates were allowed to stand for 5 min. Two Oxford cups were added with 200 μL of supernatant, and the other two were added with 200 μL of uncentrifuged bacterial culture. The plates were then incubated at 4°C for 4 h, followed by incubation at 37°C for 24 h. The inhibition diameter was then observed and recorded. Figure 5 (Table 7).

[0051] The results showed that the fermentation product of *Lactobacillus helveticus* HP-B1137 had a strong inhibitory effect on *Bacillus cereus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Proteus mirabilis*, drug-resistant *Acinetobacter baumannii*, drug-resistant *Escherichia coli*, *Salmonella typhi*, *Escherichia coli*, drug-resistant *Pseudomonas aeruginosa*, drug-resistant *Staphylococcus aureus*, and *Shigella*. Furthermore, the uncentrifuged bacterial solution had a stronger inhibitory effect than the centrifuged supernatant.

[0052] Table 7 Evaluation of the antibacterial effect of Lactobacillus helveticus HP-B1137 fermentation broth Example 8 Whole genome sequencing of Lactobacillus helveticus HP-B1137: The complete genome map of Lactobacillus helveticus HP-B1137 was obtained by sequencing and assembling using Nanopore third-generation sequencing and second-generation sequencing technology platforms. Figure 6 This is a visualized genome map of the bacterium. The genome size is 2098901 bp, with a GC ratio of 36.83%. It is a circular genome, containing 2161 CDS, 63 tRNAs, and 12 rRNAs, and includes two plasmids. Plasmid 1 is 23906 bp in size, with a GC ratio of 38.54%, and is a circular plasmid. Figure 7 As shown; plasmid 2 is 6414 bp in size, has a GC ratio of 34.49%, and is a circular plasmid, as... Figure 8 As shown.

[0053] Example 9 Genome functional annotation analysis of Lactobacillus helveticus HP-B1137 database: (1) Statistics on the proportion of annotations in general databases To obtain comprehensive gene function information, this invention performed gene function annotation using eight major databases, including UniProt, KEGG and KEGG Pathway, GO, Pfam, COG, TIGERFAMS, RefSeq, and NR. The predicted gene sequences were BLAST+ (Version: 2.11.0+) aligned with functional databases such as COG, KEGG, UniProt, and RefSeq to obtain gene function annotation results. Functional annotation was performed using the hmmer software (Version: 3.3.2) based on the Pfam and TIGERFAMS databases. Annotation statistics are shown in Table 8. Common and unique annotations for coding genes from general databases are listed below. Figure 9As shown in Table 8, all 2161 coding genes of *Lactobacillus helveticus* HP-B1137 were functionally annotated by at least one database, achieving 100% annotation coverage. The NR and Uniprot databases had the highest annotation rates (both 100%), followed by Refseq (99.26%), COG (81.58%), Pfam (78.48%), GO (73.62%), and KEGG (73.58%), indicating comprehensive and high-quality genomic functional annotation of this strain.

[0054] Table 8. Statistics of coding gene annotation results (2) Classification of Pfam structural domains The Pfam database is a homology protein family database, a collection of protein domain families, including more than 16,000 protein families. Proteins are generally composed of one or more functional regions, commonly referred to as domains. Domains appear in different combinations in different proteins, contributing to protein diversity. Identifying protein domains is crucial for understanding protein function.

[0055] This invention, based on the annotation of Pfam domains, statistically summarizes the genes annotated for each domain and plots the top 20 most annotated domains. The results are as follows: Figure 10 .Depend on Figure 10 It is known that the Pfam domain annotated in the HP-B1137 genome is distributed across multiple protein families. The first 20 major domains cover common protein families related to carbohydrate metabolism, transport, and regulation, which is consistent with the metabolic characteristics of this strain.

[0056] (3) Gene Ontology Classification Gene Ontology (GO) is an internationally standardized classification system for gene functions that provides a dynamically updated, controlled vocabulary to comprehensively describe the properties of genes and gene products in organisms.

[0057] This invention simplifies GO (Gene Ontology) annotation information to obtain GOslim classification. After summarizing and statistically analyzing gene functions from three aspects—cellular components, molecular functions, and biological processes—the top 20 most annotated GOslims under each category are selected for secondary classification and plotted. The results are as follows: Figure 11 .Depend on Figure 11It can be seen that the genes of HP-B1137 are extensively annotated in the three major GO categories of biological processes, cellular components, and molecular functions. Among them, the proportion of genes involved in metabolic processes, cellular processes, and those with catalytic activity and binding function is relatively high, reflecting the active metabolic characteristics of this strain.

[0058] (4) KEGG Pathway Classification KEGG (Kyoto Encyclopedia of Genes and Genomes) is a major public database for systematically analyzing the metabolic pathways of gene products in cells and the functions of these gene products. KEGG can be used to further study the complex biological behavior of genes.

[0059] This invention performs KEGG annotation on genes and classifies them according to the KEGG metabolic pathways they participate in. The results are as follows: Figure 12 .

[0060] (5) COG classification COG (Cluster of Orthologous Groups of Proteins) is a database for classifying gene products by orthologous origin. Each COG protein is presumed to have originated from an ancestral protein. The COG database is constructed based on the phylogenetic relationships of proteins encoded by complete genomes in bacteria, algae, and eukaryotes. By comparing Unigene with the COG database, the potential functions of genes are predicted and their functional classifications are statistically analyzed, providing a macroscopic understanding of the gene functional distribution characteristics of a species. COG is divided into 26 groups.

[0061] This invention classifies COG-annotated genes according to COG groups, and the results are as follows: Figure 13 .Depend on Figures 12-13 It is known that the HP-B1137 gene is mainly annotated to pathways such as carbohydrate metabolism, amino acid metabolism, energy metabolism and membrane transport, which is consistent with its characteristics as a lactic acid bacterium that ferments sugars to produce acid and adapts to the gastrointestinal environment.

[0062] (6) Uniprot annotation The UniProt Archive is a comprehensive, non-redundant database that contains protein sequences from all major, publicly available databases. This is because proteins may exist in different databases, and there may be multiple versions within the same database.

[0063] The number of genes annotated into the Uniprot database for the whole genome of Lactobacillus helveticus HP-B1137 described in this invention is 2161, accounting for 100.00%, indicating that all coding genes of Lactobacillus helveticus HP-B1137 can obtain protein function annotations in the Uniprot database, and the gene function annotations are complete.

[0064] (7) Refseq annotation The RefSeq (Reference Sequences) database is a collection of annotated, non-redundant transcriptomic, protein, and genomic sequences. It is a database calibrated by NCBI and other organizations, using terminology defined by the Human Genome Nomenclature Committee, and includes both official and optional gene symbols. RefSeq sequences are a non-redundant database screened by NCBI and are generally considered to have high reliability.

[0065] The number of genes annotated into the Refseq database for the whole genome of Lactobacillus helveticus HP-B1137 described in this invention is 2145, accounting for 99.26%, indicating that the vast majority of genes of Lactobacillus helveticus HP-B1137 can obtain high-reliability functional annotations from the Refseq database, further verifying the reliability of the genome annotation results.

[0066] (8) NR annotation NR Database: A non-redundant amino acid sequence database of proteins, containing non-redundant data from the SwissProt, PIR (Protein Information Resource), PRF (Protein Research Foundation), and PDB (Protein Data Bank) protein databases, as well as protein data translated from CDS data from GenBank and RefSeq.

[0067] The NR species distribution map of Lactobacillus helveticus HP-B1137 described in this invention is as follows: Figure 14 .Depend on Figure 14 It can be seen that the sequences homologous to the HP-B1137 encoding gene mainly come from lactic acid bacteria groups such as Lactobacillus, which is consistent with its taxonomic status identified based on 16S rRNA.

[0068] (9) TIGRFAMs annotations TIGRFAMs is a database of multiple sequence alignments, hidden Markov models (HMMs), and related information. It is a member of interPro and is used for protein sequence classification, supporting automatic annotation (mostly prokaryotic) proteins. Starting with v10.0, the TIGRFAMs model uses HMM 3, which offers excellent retrieval speed and sensitivity.

[0069] The number of genes annotated into the Tigrfams database for the whole genome of Lactobacillus helveticus HP-B1137 described in this invention is 1064, accounting for 49.24%. This indicates that nearly half of the genes of Lactobacillus helveticus HP-B1137 can be classified into the known TIGRFAMs protein family, and the remaining genes may encode functional proteins unique to this strain.

[0070] Example 10 Notes from the proprietary database of Lactobacillus helveticus HP-B1137: This invention provides personalized analysis of Lactobacillus helveticus HP-B1137, including annotation of it using proprietary databases such as ARDB database annotation, CAZy database-based carbohydrate enzyme annotation, pathogen-host interaction analysis, drug resistance gene annotation, cytochrome P450 annotation, VFDB annotation, signal peptide prediction, type III secretory system effector protein annotation, and TCDB transporter protein annotation.

[0071] The complete genome of *Lactobacillus helveticus* HP-B1137 described in this invention, after annotation in proprietary databases, revealed the following: no resistance genes were annotated in the ARDB database; 763 perfectly matched gene sequences were annotated in the pathogen-host interaction database; no drug resistance genes were annotated in the CARD database; 412 perfectly matched virulence factors were annotated in the VFDB database; 161 perfectly matched gene sequences were annotated in the type III secretory system effector protein database; carbohydrate enzyme annotation results are shown in Table 9; predicted signal peptides of the *Lactobacillus helveticus* HP-B1137 genome are shown in Table 10; predicted transmembrane and secretory proteins are shown in Table 11; cytochrome P450 annotation results are shown in Table 12; and TCDB membrane transport protein annotation results are shown in Table 13. Figure 15 In summary, no drug resistance genes were annotated for Lactobacillus helveticus HP-B1137, indicating good safety. CAZy annotation shows that it contains 29 glycoside hydrolase (GH) encoding genes, which is consistent with its ability to hydrolyze isoflavone glucoside to isoflavone, providing genomic evidence for elucidating its biotransformation mechanism.

[0072] Table 9 CAZy Notes Table 10 Statistical Table of Signal Peptide Prediction Table 11 Predictive Statistics of Transmembrane Proteins and Secretory Proteins Table 12 Cytochrome P450 Notes Example 11 The ability of Lactobacillus helveticus HP-B1137 to convert verbascoside glucoside to verbascoside: A single colony of *Lactobacillus helveticus* HP-B1137 from MRS solid medium was inoculated onto 30 mL of freshly sterilized MRS liquid medium using a bamboo stick and cultured anaerobically at 37°C and 180 rpm for 6 h. Verbena isoflavone glucoside was added to the above MRS liquid medium at a concentration of 0.2 mmol / L and cultured anaerobically at 37°C and 180 rpm for 20 h. The fermentation broth and extraction solvent (ethyl acetate) were added to a separatory funnel and vortexed to mix. The bacterial culture was centrifuged at 3580 rpm for 10 min, and the supernatant was collected, rotary evaporated at 55°C, dissolved in 10% acetonitrile, and subjected to high-performance liquid chromatography (HPLC).

[0073] Inverted C 18 The high-performance liquid chromatography (HPLC) conditions for the column are shown in Tables 13 and 14: Table 13 Column Equilibrium System Table 14 Gradient elution program (C: Acetonitrile, D: Wahaha water) Using 10% acetonitrile as the mobile phase, 5 μL was injected, and the chromatogram at 254 nm was analyzed. The results are as follows: Figure 16 As shown. Using the content of verbenafil isoflavone glucoside before fermentation as an indicator, after 12h, 24h and 36h of conversion, it was found that the content of verbenafil isoflavone increased with the increase of time, and at 36h, verbenafil isoflavone glucoside had been completely converted into verbenafil isoflavone.

[0074] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Lactobacillus helveticus ( Lactobacillus helveticus HP-B1137, characterized in that, The Lactobacillus helveticus HP-B1137 was deposited at the China Center for Type Culture Collection (CCTCC) on July 30, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261780.

2. A microbial preparation, characterized in that, Includes Lactobacillus helveticus HP-B1137 as described in claim 1.

3. The application of *Lactobacillus helveticus* HP-B1137 as described in claim 1 or the microbial preparation as described in claim 2 in the preparation of antibacterial products, characterized in that... The antibacterial product is used to inhibit at least one of Bacillus cereus, Klebsiella pneumoniae, Acinetobacter baumannii, Proteus mirabilis, Salmonella typhi, Escherichia coli, drug-resistant Pseudomonas aeruginosa, drug-resistant Staphylococcus aureus, and Shigella.

4. An antibacterial product, characterized in that, Includes Lactobacillus helveticus HP-B1137 as described in claim 1 or the microbial preparation as described in claim 2.

5. The antibacterial product according to claim 4, characterized in that, The antibacterial product is used to inhibit at least one of Bacillus cereus, Klebsiella pneumoniae, Acinetobacter baumannii, Proteus mirabilis, drug-resistant Acinetobacter baumannii, drug-resistant Escherichia coli, Salmonella typhi, Escherichia coli, drug-resistant Pseudomonas aeruginosa, drug-resistant Staphylococcus aureus, and Shigella.

6. The use of Lactobacillus helveticus HP-B1137 as described in claim 1 or the microbial preparation as described in claim 2 in the conversion of verbascoside glucoside into verbascoside.

7. A method for preparing verbascoside isoflavone, characterized in that, The process includes the following steps: mixing Lactobacillus helveticus HP-B1137 as described in claim 1 or the microbial preparation as described in claim 2 with vernix caseosa glucoside, anaerobic culture, and then isolating vernix caseosa from the fermentation product after the culture is completed.

8. The preparation method according to claim 7, characterized in that, The anaerobic culture method is as follows: add the verrucous isoflavone glucoside to the culture medium of Lactobacillus helveticus HP-B1137 and culture anaerobically at 180 r / min for 24-36 h.

9. The preparation method according to claim 8, characterized in that, The concentration of the verbascoside glucoside added to the culture medium was 0.2 mmol / L.

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