Lactobacillus reuteri GY-18 and application thereof in increasing astragaloside content

Abstract

The invention provides lactobacillus reuteri GY-18 and application thereof in increasing the content of astragaloside, and belongs to the technical field of microorganisms, fermentation and natural product biotransformation. The lactobacillus reuteri GY-18 capable of improving the content of astragaloside in a fermented astragalus membranaceus product is obtained through screening, the lactobacillus reuteri GY-18 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC M 20252796, and the lactobacillus reuteri GY-18 can be used for improving the content of astragaloside in the fermented astragalus membranaceus product. The lactobacillus reuteri GY-18 can secrete acetylxylan esterase for promoting conversion of astragaloside into astragaloside IV, has efficient specificity on conversion of astragaloside into astragaloside IV, and is high in conversion capacity; according to the method, the content of astragaloside IV is remarkably increased within 24 hours in a semi-solid state fermentation mode, and the method has good practicability.

Description

Technical Field

[0001] This invention belongs to the field of microbial, fermentation and natural product biotransformation technology, specifically relating to a type of Lactobacillus reuteri GY-18 and its application in increasing the content of astragaloside A. Background Technology

[0002] Astragalus is a traditional bulk medicinal herb. Its pharmacologically active components, particularly astragaloside A, possess various pharmacological activities, including anti-inflammatory, immunomodulatory, and cardiovascular protective effects. However, the large molecular weight and strong hydrophilicity of astragaloside A result in low oral bioavailability, severely limiting its clinical application. Therefore, the 2020 edition of the Chinese Pharmacopoeia has listed astragaloside A content as a key indicator for evaluating the quality of astragalus. To improve the yield and bioavailability of astragaloside A, microbial fermentation has been extensively studied.

[0003] Existing technologies mostly employ filamentous fungi (such as Aspergillus niger, Poria cocos, and Trametes versicolor) or certain bacteria to ferment Astragalus membranaceus, which can increase the content of astragaloside A to a certain extent. However, these methods have the following obvious limitations: (1) Fungal fermentation usually takes more than 5-10 days, with a long fermentation cycle and low production efficiency; (2) Bacterial fermentation mostly adopts liquid deep fermentation, and the subsequent cell separation and extraction steps are cumbersome, energy consumption is high, and it is not conducive to large-scale production; (3) The strains used are mostly non-food grade microorganisms, which may produce toxins or other unknown secondary metabolites, making it difficult to guarantee the safety of the final product; (4) The microbial transformation pathway is unclear, the mechanism is not well understood, and it is impossible to achieve targeted regulation, resulting in poor product repeatability and unstable quality. In contrast, lactic acid bacteria, as a recognized safe food grade microorganism, have been widely used in the food industry. If a food grade lactic acid bacterium that can efficiently and specifically convert astragaloside A into astragaloside A can be obtained, and a simple and rapid fermentation process is established to match it, it will have important industrial value. However, to date, there are no systematic reports on significantly increasing astragaloside A content using specific lactic acid bacteria, especially food-grade strains. Therefore, it is necessary to screen for a strain that can increase astragaloside A content. Summary of the Invention

[0004] To address some shortcomings in existing technologies, this invention provides *Lactobacillus reuteri* GY-18 and its application in increasing the content of astragaloside A. This invention screened and obtained a strain of *Lactobacillus reuteri* GY-18 capable of increasing the content of astragaloside A in fermented astragalus products. *Lactobacillus reuteri* GY-18 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252796. *Lactobacillus reuteri* GY-18 secretes acetylxylan esterase, which promotes the conversion of astragaloside saponins to astragaloside A, exhibiting high efficiency and specificity in this conversion and strong conversion ability. This invention achieves a significant increase in astragaloside A content within 24 hours using a semi-solid-state fermentation method, demonstrating excellent practicality.

[0005] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0006] This invention first provides an application of acetylxylan esterase, the amino acid sequence of which is shown in SEQ ID NO:1, and the application includes any of the following:

[0007] (1) Catalyzes the conversion of astragaloside saponins into astragaloside A;

[0008] (2) Increase the content of astragaloside A in products containing astragalus;

[0009] (3) Prepare food or health products with high astragaloside A content.

[0010] This invention also provides applications of biomaterials, wherein the biomaterials are: polynucleotides, expression cassettes, and cells;

[0011] The polynucleotide encodes an acetylxylan esterase with an amino acid sequence as shown in SEQ ID NO:1;

[0012] The expression cassette contains the polynucleotide;

[0013] The cells are cells carrying the polynucleotide or expression cassette, or containing the vector, or capable of expressing acetylxylan esterase as shown in SEQ ID NO:1, or recombinant cells;

[0014] The application includes any of the following:

[0015] (1) Catalyzes the conversion of astragaloside saponins into astragaloside A;

[0016] (2) Increase the content of astragaloside A in products containing astragalus;

[0017] (3) Prepare food or health products with high astragaloside A content.

[0018] The present invention also provides a *Lactobacillus reutrei* GY-18, wherein the *Lactobacillus reutrei* GY-18 expresses an acetylxylan esterase with the amino acid sequence shown in SEQ ID NO:1; the *Lactobacillus reutrei* GY-18 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252796, deposit date December 8, 2025, deposit address Wuhan, China, and classification name: *Linosilactobacillus reutrei* GY-18.

[0019] The present invention also provides a microbial agent comprising the aforementioned *Lactobacillus reuteri* GY-18.

[0020] The present invention also provides the application of the above-mentioned *Lactobacillus reuteri* GY-18 or the above-mentioned microbial agent, wherein the application includes any of the following:

[0021] (1) Catalyzes the conversion of astragaloside saponins into astragaloside A;

[0022] (2) Increase the content of astragaloside A in products containing astragalus;

[0023] (3) Prepare food or health products with high astragaloside A content.

[0024] This invention also provides a method for catalyzing the conversion of astragaloside saponins to astragaloside A, the method comprising:

[0025] Add acetyl xylan esterase, or the above-mentioned Lactobacillus reuteri GY-18, or the above-mentioned microbial agent to the culture medium or raw material containing astragaloside.

[0026] Preferably, the method involves activating Lactobacillus reuteri GY-18, then mixing the activated seed liquid with Astragalus membranaceus raw material for semi-solid fermentation.

[0027] Preferably, the method includes:

[0028] (1) Activate Lactobacillus reuteri GY-18 to obtain activated seed liquid;

[0029] Astragalus raw materials are dried, pulverized, and sterilized to obtain Astragalus powder;

[0030] (2) Centrifuge the obtained seed liquid to collect the bacterial cells, resuspend them in sterile physiological saline, and mix the resuspended bacterial suspension with Astragalus powder at a mass ratio of 1:1 to form a semi-solid fermentation substrate.

[0031] (3) The obtained semi-solid fermentation substrate was statically cultured at 37℃ for 18-36h to convert astragaloside into astragaloside A.

[0032] Preferably, in step (1), the OD value of the seed liquid is 0.8-1.0.

[0033] Preferably, in step (2), the bacterial suspension and Astragalus powder are in a mass ratio of 1:1; the OD value of the bacterial suspension is 0.5-0.6.

[0034] A pH buffer was also added to the semi-solid fermentation substrate.

[0035] More preferably, the pH buffer comprises 0.1%-5% (w / w, based on the mass of Astragalus powder) of calcium carbonate.

[0036] Preferably, in step (3), the culture is incubated at 37°C for 24 hours.

[0037] The present invention also provides a food with high astragaloside A content, which is prepared by the above method, and the astragaloside A content in the food is increased by more than 50% compared with unfermented astragalus raw material.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] (1) The present invention screened and obtained a strain of Lactobacillus reuteri GY-18, which is a recognized safe food-grade lactic acid bacterium, non-pathogenic, safe and reliable. Studies have shown that this strain can efficiently and specifically convert astragaloside into astragaloside A, with strong conversion ability.

[0040] (2) This invention employs a semi-solid-state fermentation method to ferment Astragalus membranaceus raw materials, achieving the conversion of astragaloside to astragaloside A. The method is simple, requiring no complex liquid fermentation tanks or subsequent solid-liquid separation steps, resulting in low energy consumption and easy scale-up production. Its production complexity is lower than existing bacterial liquid fermentation methods. The semi-solid-state fermentation method of this invention has an extremely short fermentation cycle, achieving significant conversion results in just 24 hours, with production efficiency far exceeding the 5-10 days required by existing fungal fermentation technologies.

[0041] (3) This invention is the first to identify acetylated xylan esterase (AXE) in *Lactobacillus reuteri* as a key enzyme catalyzing the deacetylation of astragaloside to astragaloside A. By regulating the expression of the key enzyme and optimizing the fermentation process, targeted control of the conversion process can be achieved, ensuring the stability and reproducibility of product quality.

[0042] (4) The Astragalus product fermented by the method described in this invention has a high astragaloside A content. Compared with unfermented Astragalus raw materials, the astragaloside A content of the product described in this invention can be increased by more than 50%. The fermentation method described in this invention only requires 24 hours to increase the astragaloside A content by 60%-75%, demonstrating excellent industrialization capabilities. Furthermore, the fermented product can be directly used as a raw material for developing foods and health products with functions such as enhancing immunity, protecting the cardiovascular system, and anti-aging, resulting in high added value and excellent practicality. Attached Figure Description

[0043] Figure 1 This is a comparison of the high-performance liquid chromatograms of Astragalus membranaceus raw materials detected by the method described in the pharmacopoeia (top figure) and the method described in this invention (bottom figure).

[0044] Figure 2 is Phylogenetic analysis of 45 strains that initially showed positive conversion ability of astragaloside A and the increase rate of astragaloside A content after fermentation of astragalus; the numbers in parentheses after the strain number in the figure represent the percentage increase in astragaloside A content.

[0045] Figure 3 The effect of different culture times (A) and calcium carbonate addition (B) on astragaloside A content under semi-solid fermentation conditions of Lactobacillus reuteri GY-18; different lowercase letters in the figure represent differences that reached the statistical significance level (P<0.05).

[0046] Figure 4 The figures show the relative expression levels of the acetylxylan esterase (AXE) and acetylesterase (ACE) genes in Lactobacillus reuteri GY-18 induced by total saponins from Astragalus membranaceus; in the figure, ** indicates that the difference reached a highly significant level (P<0.01). Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the embodiments of the present invention, unless otherwise described, conventional experimental methods are used. The processes involved in the embodiments, unless otherwise described, are those that can be understood and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0048] The astragaloside A detection system used in the following examples is as follows:

[0049] Extraction method: Take Astragalus membranaceus powder (sources: Astragalus membranaceus from Hengshan, Datong, Shanxi; Astragalus membranaceus from Zizhou, Yulin, Shaanxi; wild Astragalus membranaceus from Minxian, Dingxi, Gansu; wild Astragalus membranaceus from Guyang and Ming'an, Inner Mongolia; wild Astragalus membranaceus from Yichun, Heilongjiang, Northeast China, all of which are commercially available), add 80% methanol solution containing 4% concentrated ammonia test solution, heat under reflux for one hour, filter and evaporate the filtrate to dryness, dilute the residue with 80% methanol solution to volume, and filter to obtain the test solution.

[0050] Detection conditions (optimized): Column: C18 (250mm×4.6mm, 5μm); Mobile phase A: acetonitrile, Mobile phase B: ultrapure water + 0.1% (v / v) formic acid (A:B 32:68); Flow rate: 1mL / min; Column temperature: 26℃; Detector: evaporative light scattering detector; Injection volume: 10μL.

[0051] This detection system, compared to the one described in the Chinese Pharmacopoeia, adds 0.1% formic acid to mobile phase B. The results of the two detections are as follows: Figure 1 As shown in the figure, the baseline drift and poor peak resolution of the liquid chromatogram under the original detection conditions in the pharmacopoeia resulted in the measured value being nearly 10 times higher than the actual value. Figure 1 Through system debugging, adding 0.1% formic acid to the mobile phase effectively suppressed peak tailing and improved separation performance. Figure 1 The optimized detection method features sharp peaks and a stable baseline, enabling accurate determination of astragaloside A content in raw materials and fermentation samples.

[0052] Therefore, the following examples use optimized detection conditions to detect the astragaloside A content in raw materials and fermentation samples.

[0053] In the following examples, the formulation of MRS liquid culture medium containing 0.05% (w / v) total astragalus saponins (Shanghai Yuanye) was as follows: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g K₂HPO₄, 2.0 g triammonium citrate, 5.0 g sodium acetate, 0.2 g MgSO₄·7H₂O, 0.05 g MnSO₄·H₂O, 0.5 g total astragalus saponins (purity ≥95%), and 1.08 g Tween-80. The solution was dissolved in distilled water and brought to a final volume of 1 L. The pH was adjusted to 6.4 with NaOH or HCl, and the medium was sterilized at 121°C for 15 minutes before use.

[0054] The formulation of heat-treated nutrient broth liquid culture medium containing 0.05% (w / v) total saponins of Astragalus membranaceus (Shanghai Yuanye) is as follows: 5.0 g peptone, 3.0 g beef extract, and 5.0 g NaCl are dissolved in distilled water and brought to a final volume of 1 L. The pH is adjusted to 7.0 with NaOH or HCl, and the medium is sterilized at 121°C for 15 minutes before use. Simultaneously, the samples must be heat-treated at 80°C for 10 minutes before inoculation and culture.

[0055] In both liquid culture media, the total saponins of Astragalus membranaceus can be prepared into a stock solution of 100 g / L. After being sterilized by filtration through a 0.22 μm filter membrane, the solution is aseptically added after the culture medium has been sterilized and cooled.

[0056] The formula for MRS liquid culture medium is as follows: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g K₂HPO₄, 2.0 g triammonium citrate, 5.0 g sodium acetate, 0.2 g MgSO₄·7H₂O, 0.05 g MnSO₄·H₂O, and 1.08 g Tween-80. Dissolve in distilled water and bring the volume to 1 L. Adjust the pH to 6.4 with NaOH or HCl, and sterilize at 121°C for 15 minutes. Example 1: Screening and identification of highly efficient transforming strains.

[0057] S1. Sample enrichment and initial screening:

[0058] Twenty samples of Astragalus membranaceus from five producing areas—Shanxi, Shaanxi, Gansu, Inner Mongolia, and Northeast China—were collected and labeled as follows: (Source: Astragalus membranaceus from Hengshan Mountain in Datong, Shanxi; Astragalus membranaceus from Zizhou in Yulin, Shaanxi; Wild Astragalus membranaceus from Minxian County in Dingxi, Gansu; Wild Astragalus membranaceus from Guyang and Ming'an in Inner Mongolia; Wild Astragalus membranaceus from Yichun in Heilongjiang, Northeast China; all commercially available). These samples were then pulverized to obtain Astragalus membranaceus powder from different sources.

[0059] 5.0 g of Astragalus powder from different sources was weighed and added to 5.0 mL of sterile physiological saline. After mixing, the samples were incubated at 30 °C for 48 hours for enrichment. After the incubation period, the content of astragaloside A in each enriched sample was determined by high performance liquid chromatography (HPLC) under optimized conditions.

[0060] The test results showed that after enrichment, the astragaloside A content of one sample (GY) from Guyang, Inner Mongolia and one sample (MA) from Ming'an, Inner Mongolia exceeded 160% of that of the untreated raw material. These two samples were then marked as target samples.

[0061] S2. Strain Isolation and Purification:

[0062] Considering the unique taxonomic characteristics of food-grade bacterial strains, this step selectively isolates bacteria from the Lactobacilliles (where lactic acid bacteria belong), the Bacillus subtilis, and the Bacillus coagulans (where Bacillus coagulans belong). The specific steps are as follows:

[0063] S21. Selective isolation of bacterial communities in target sample GY:

[0064] Add 9.0 mL of sterile physiological saline to 1.0 g of the target sample numbered GY, and vortex to mix thoroughly to prepare 10 -1The bacterial suspension was diluted to a certain concentration, and then serially diluted 10-fold. 100 μL of each of the 10-fold dilutions was used. -4 10 -5 10 -6 The diluted bacterial suspensions were spread on R2A solid medium plates (formulation: 0.5g yeast extract, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.024g anhydrous magnesium sulfate, 0.3g sodium pyruvate, 15.0g agar, pH adjusted to 7.2±0.2), with each dilution performed in triplicate.

[0065] The coated plates were inverted and incubated in a 30℃ constant temperature incubator for 5 days. After the incubation period, clear single colonies grew on the plates. Based on the differences in colony morphology and color, a total of 110 single colonies were picked from plates of appropriate dilution from the GY sample. These colonies were inoculated into MARS liquid medium containing 0.05% (w / v) astragalus total saponins (Shanghai Yuanye) for Lactobacillus and heat-treated nutrient broth liquid medium containing 0.05% (w / v) astragalus total saponins (Shanghai Yuanye) for Bacillus, respectively. The media were incubated at 30℃ for 48 hours. After the incubation period, the culture medium was collected, and the content of astragaloside A was detected according to the optimized method.

[0066] The results showed that a total of 110 strains of bacteria were isolated from the target sample numbered GY. In MRS liquid medium or heat-treated nutrient broth liquid medium, 23 strains of bacteria grew and astragaloside A was detected. Among them, 18 strains were from the Bacillus order and 5 strains were from the Lactobacillus order, indicating that these strains have the potential to convert the total astragaloside precursor added to the culture medium into astragaloside A. Further screening was conducted on these strains.

[0067] S22. Selective isolation of bacterial communities in target sample MA:

[0068] Following the procedure in step S21, selective isolation of the bacterial community in the target sample numbered MA was performed. The results showed that a total of 103 bacterial strains were isolated from the target sample numbered MA. In MRS liquid medium or heat-treated nutrient broth liquid medium, 22 bacterial strains grew and astragaloside A was detected. Among them, 16 strains belonged to the Bacillus order and 6 strains belonged to the Lactobacillus order, indicating that these strains have the potential to convert the astragaloside A precursor added to the culture medium into astragaloside A. Further screening was conducted on these strains.

[0069] S3. Strain identification and rescreening:

[0070] The strains that detected astragaloside A in the target sample numbered GY and the strains that detected astragaloside A in the target sample numbered MA in step S2 were purified by streaking on the corresponding MRS agar plates or heat-treated nutrient broth agar plates, resulting in a total of 45 strains.

[0071] After purification, genomic DNA was extracted from single colonies. 16S rRNA gene amplification was performed using the universal bacterial primer 27F / 1492R, followed by sequencing. The sequencing results were compared using BLAST in the NCBI database to determine the bacterial species classification. The identification results showed that the 45 strains belonged to 14 species in 6 genera within the orders Bacillus and Lactobacillus. Figure 2) .

[0072] The sequence of the bacterial universal primer 27F / 1492R is as follows:

[0073] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 5);

[0074] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 6).

[0075] To verify the ability of these strains to directly ferment Astragalus membranaceus raw materials, this step involves a semi-solid fermentation re-screening. The specific steps are as follows:

[0076] (a) Each purified strain was inoculated into MRS liquid medium for activation and cultured until OD500. 600 The value is approximately 0.8-1.0. Then, take 2 mL of bacterial suspension, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, and resuspend the bacterial cells in 5 mL of sterile physiological saline. The OD value of the resuspended bacterial suspension is... 600 The value is 0.5-0.6.

[0077] (b) The resuspended bacterial solution was thoroughly mixed with 5g of sterile Astragalus powder (121°C, 20 minutes) in a sterile Erlenmeyer flask to form a semi-solid fermentation system.

[0078] (c) The obtained semi-solid fermentation system was placed in a 37℃ incubator for static fermentation for 24 hours. After fermentation, samples were taken to detect the astragaloside A content, and the increase rate relative to the uninoculated sterilized astragalus powder control group was calculated. The strain classification of the 45 strains and their transformation astragaloside A content increase rates are as follows: Figure 2 Show.

[0079] from Figure 2 is available.It can be seen that the astragaloside A content increased by 15% to 56% after transformation by 45 strains. Among them, the strain identified as Limosilactobacillus reuteri, numbered GY-18, performed the best, with the astragaloside A content increasing by 56% after 24 hours of fermentation.

[0080] The *Limosilactobacillus reuteri* GY-18 strain was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252796, deposit date December 8, 2025, address in Wuhan, China, and classification name: *Linosilactobacillus reutrei* GY-18.

[0081] Example 2:

[0082] This embodiment analyzes the mechanism by which *Lactobacillus reuteri* GY-18 can increase the content of astragaloside A, predicts and mines the key enzymes involved, and verifies their expression. The specific steps are as follows:

[0083] (1) Prediction and gene mining of key enzymes:

[0084] Based on the analysis of the chemical structure of astragaloside, the key step in its conversion to astragaloside A is the removal of the acetyl group on the xylose residue. This reaction may be catalyzed by deacetylase. Based on the chemical structure analysis and enzymatic mechanism inference of astragaloside, this step focuses on the deacetylases acetylxylan esterase (AXE, belonging to the carbohydrate esterase family), acetyl esterase (ACE), and peptidoglycan deacetylase (PGD).

[0085] Whole-genome sequencing was performed on *Lactobacillus reuteri* GY-18 (Beijing Novogene Technology Co., Ltd.). The amino acid sequences of the reported model enzymes AXE, ACE, and PGD were compared locally using the whole-genome predicted protein database for GY-18. The results revealed a coding gene in the GY-18 genome with 26.8% similarity (E-value < 1e-5) to the known AXE sequence from *Neocallimastixpatriciarum* (GenBank: AAB69091.1), named axe_GY18. Simultaneously, a coding gene with 28.6% similarity (E-value < 1e-5) to the known ACE sequence from *Lactobacillus acidophilus* (GenBank: AAV42042.1) was also found, named ace_GY18.

[0086] The inferred coding sequences of these two genes are as follows:

[0087] The protein sequence encoded by the axe_GY18 gene (SEQ ID NO:1):

[0088] MKKYQPTDLIKFASRTGRWTVKKINNVPTLYTTNLGSYLRFKISNAKKCQITVLPNQNSLSPSQVFAFRIDGGKWQRAQASLEKIDIPLDSTLHTIEIMAAGNTDIDEVWQGNEGFAIKNIYLDNGEIMATPQRPVVNFIGDSITAGCWVVGNHPAADYRP ETNYAGICADLLNVDSVRIAYSAGGVLRPATGGVPTADVFLGKIDAQTSWTPNHPDLNVINLGVNDRRFPLAQFTAAFDLFIQQVKLTFPHTPLAIMIPFSQTFASEIRKIAIKHKCSIIETKTWHHSFTDGLHPDQAGAIAEGKMLAQALQPLSPQFSVQ;

[0089] Nucleotide sequence of the axe_GY18 gene (SEQ ID NO:2):

[0090] ATGAAGAAATATCAACCAACCGATCTAATTAAATTTGCTTCACGTACTGGACGCTGGACCGTTAAAAAGATTAATAATGTGCCCACCTTATACACCACTAACCTTGGCAGCTACCTTCGTTTTAAGATTTCAAATGCTAAAAAATGTCAAATTACTGTTTTGCCAAATCAAAATTCCCTATCTCCTAGCCAAGTGTTTGCTTTTCGAATTGATGGCGGAAAGTGGCAACGGGCACAAGCTAGCTTAGAAAAAATTGATATTCCACTTGATTCGACATTACATACCATCGAAATAATGGCAGCCGGTAATACCGATATTGACGAAGTGTGGCAAGGCAATGAAGGCTTTGCAATTAAAAATATTTATCTCGATAACGGCGAAATTATGGCAACCCCGCAGCGTCCTGTGGTCAACTTTATCGGTGATTCAATCACGGCCGGTTGTTGGGTAGTTGGCAATCACCCTGCCGCTGACTACCGACCAGAAACTAACTATGCGGGAATCTGTGCAGACCTGCTTAACGTCGATAGTGTTCGAATAGCCTACTCTGCTGGAGGAGTGCTAAGACCTGCTACTGGCGGGGTACCAACTGCCGATGTCTTTTTAGGAAAAATTGATGCTCAAACATCATGGACGCCTAATCATCCTGATCTCAACGTGATTAATTTAGGCGTTAATGACCGTCGATTCCCCCTAGCTCAATTTACTGCCGCTTTCGACCTTTTTATCCAGCAAGTTAAGCTAACCTTTCCACATACTCCTTTAGCGATCATGATTCCTTTTAGTCAGACTTTTGCTTCTGAGATTCGAAAAATCGCCATTAAACATAAATGTTCGATAATTGAAACCAAAACTTGGCACCATAGTTTCACTGATGGGCTCCATCCTGATCAAGCTGGCGCTATCGCGGAAGGAAAAATGCTTGCGCAAGCTTTACAACCTCTTTCACCTCAATTCAGTGTACAATAG;

[0091] The protein sequence encoded by the ace_GY18 gene (SEQ ID NO:3):

[0092] MEIKSVNLDQPYSSLDIYHSNTDKALPGLVILPGGSYNQIMERDSERVALTFATHAWQTFVVRYPVVEHKNYEEAKIAVHQAFEYIVNHADKLDVDADRLGIIGFSAGGQIAAAYSNEK LTHAKFAALGYPVIQPLIDERMGVTTENVAELVNPQTPPTFMWGSAKDELTPFVDHLQVYADALIKNNVPYELHEFGTGGHGIALANKYTGIVNNDRVDTHMGKWFSLFLEWLTELNLI;

[0093] Nucleotide sequence of the ace_GY18 gene (SEQ ID NO:4):

[0094] .

[0095] (2) Validation of the expression of key enzymes:

[0096] To further determine which of the two enzymes obtained in step (1) plays a dominant role in the transformation of astragaloside, specific primers were designed for this step. The expression levels of the two genes under induction conditions were detected by real-time quantitative PCR (RT-qPCR). The specific steps are as follows:

[0097] RNA extraction: GY-18 strain was inoculated into two liquid media: (A) MRS liquid medium; (B) MRS medium containing 0.05% (w / v) total astragalus saponins. After inoculation, the culture media were incubated statically at 37°C until mid-log phase (approximately 12 hours). Then, equal amounts of bacterial cells were taken from both media, and total RNA was extracted using an RNA extraction kit (TIANGEN, DP430). Genomic DNA contamination was removed by treatment with DNase I.

[0098] cDNA synthesis: 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara RR047A) to obtain the bacterial cDNA template.

[0099] RT-qPCR: The axe_GY18 and ace_GY18 genes were detected by real-time quantitative PCR (RT-qPCR) using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, AG11701) on a QuantStudio series real-time PCR instrument.

[0100] Reaction system (20 μL): 2 μL bacterial cDNA template, 0.4 μL each of forward and reverse primers (10 μM), 10 μL SYBR Green Mix, and ddH2O to a final volume of 20 μL.

[0101] Reaction procedure: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds, annealing / extension at 60℃ for 30-34 seconds, 40 cycles; finally, melting curve analysis was performed. The 16S rRNA gene of GY-18 was used as an internal reference gene.

[0102] The upstream and downstream primer sequences are as follows:

[0103] ax_GY18-F:5'-GGATCTTCCAGAGATATGCAACTATTCGATCTGCCG-3'(SEQ ID NO:7)

[0104] ax_GY18-R:5'-CTGCCGTTCGACGATTCAGCCTTTAAGATGCTGCTTAAA-3'(SEQ ID NO:8)

[0105] ace_GY18-F:5'-GGATCTTCCAGAGATGCACTACTTACTGGTGCATGGAAA-3'(SEQ ID NO:9)

[0106] ace_GY18-R:5'-CTGCCGTTCGACGATCTAAATTAAGTTCAATTCAGTTAACCACTC-3'(SEQ IDNO:10)

[0107] Test results as follows Figure 4 As shown.

[0108] from Figure 4 As can be seen, compared with growth in basal MRS medium, the expression level of the axe_GY18 gene was significantly upregulated when GY-18 was grown in medium containing total astragalus saponins, approximately 6.72 times that of the control group (P<0.01). However, the expression level of the ace_GY18 gene was only slightly upregulated (approximately 1.38 times), and the difference was not significant (P>0.05). This indicates that acetylated xylan esterase (AXE) is an enzyme that responds to astragalus saponin induction and may play a key role in the transformation process.

[0109] (3) Bioinformatics analysis of acetylated xylan esterase (AXE_GY18):

[0110] This step predicts the properties of the AXE_GY18 protein, whose amino acid sequence is shown in SEQ ID NO:1. The specific results are as follows:

[0111] Basic physicochemical properties: Using the ExPASy ProtParam tool, its theoretical molecular weight is predicted to be 35.51 kDa, and its theoretical isoelectric point (pI) is 8.59, classifying it as a basic protein. Instability index prediction indicates that this protein is stable.

[0112] Secondary structure: Using the SOPMA tool, the secondary structure of this protein was predicted to consist of 19.25% α-helices, 25.78% extended strands, 0% β-turns, and 54.97% random coils. The abundant random coil regions may be related to its substrate binding and catalytic flexibility.

[0113] Hydrophilicity / hydrophobicity: Analysis using the ExPASy ProtScale tool (based on the Kyte-Doolittle algorithm) showed that the protein is generally hydrophobic.

[0114] Signal peptide and transmembrane region: Using online tools TMHMM 2.0 and SignalP 6.0, the protein sequence does not contain typical transmembrane helical domains and signal peptide sequences. This suggests that AXE_GY18 is likely an intracellular protein, consistent with the high transformation activity of the bacterial lysate in Example 1.

[0115] Example 3: Optimization of fermentation process for Lactobacillus reuteri GY-18

[0116] This invention optimizes the semi-solid fermentation conditions of *Lactobacillus reuteri* GY-18 screened in Example 1 by adjusting fermentation time and pH buffering. The specific steps are as follows:

[0117] The steps for the semi-solid fermentation of *Lactobacillus reuteri* GY-18 are as follows:

[0118] (a) Each purified strain was inoculated into MRS liquid medium for activation and cultured until OD500. 600 The value was 0.8-1.0. Then, 2 mL of bacterial suspension was taken, centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, and the bacterial cells were resuspended in 5 mL of sterile physiological saline. The OD value of the resuspended bacterial suspension was... 600 The value is 0.5-0.6.

[0119] (b) The resuspended bacterial solution was thoroughly mixed with 5g of sterile Astragalus powder (121°C, 20 minutes) in a sterile Erlenmeyer flask to form a semi-solid fermentation system.

[0120] (c) The obtained semi-solid fermentation system was placed in a 37℃ incubator for static fermentation. After the fermentation was completed, samples were taken to detect the content of astragaloside A.

[0121] (1) Optimization of fermentation time:

[0122] Fermentation times were set to 12h, 24h, 36h, and 48h respectively. After fermentation, samples were taken to test the astragaloside A content. The results are as follows: Figure 3 As shown in Figure A.

[0123] As shown in the figure, under semi-solid fermentation conditions with a bacterial suspension to astragalus powder mass ratio of 1:1, the astragaloside A content reached 0.920 mg / g after 24 hours of fermentation, an increase of 65.5% compared to the control of 0.56 mg / g. After 36 and 48 hours of fermentation, the astragaloside A content reached 0.960 mg / g and 0.977 mg / g, respectively, still increasing but showing no significant difference from the 24-hour fermentation level. Figure 3 A). Considering production efficiency, 24 hours is preferred as the fermentation endpoint.

[0124] (2) Optimization of pH buffer:

[0125] Lactic acid bacteria production may inhibit bacterial activity and product stability. Therefore, in this step, different proportions (0%, 1%, 3%, 5%, based on the mass w / w of Astragalus powder) of calcium carbonate were added to the semi-solid fermentation system obtained in step (b) of the semi-solid fermentation. The resulting semi-solid fermentation system was then placed in a 37℃ incubator for static fermentation for 24 hours. After fermentation, samples were taken to detect the astragaloside A content. The results are as follows: Figure 3 As shown in B.

[0126] As can be seen from the figure, adding 3% calcium carbonate can effectively stabilize the pH of the fermentation system at around 6.0. At the same time, it can increase the astragaloside A content in the fermented product to the highest level of 0.973 mg / g after 24 hours of fermentation, which is higher than 0.927 mg / g with 1% calcium carbonate and 0.956 mg / g with 5% calcium carbonate. It is 73.8% higher than the control of 0.56 mg / g, and is close to the level when no buffer is added and fermentation is carried out for 48 hours.

[0127] In summary, under the premise that the physiological saline bacterial suspension of Lactobacillus reuteri GY-18 (OD value of 0.5-0.6) and sterilized Astragalus powder are mixed at a mass ratio of 1:1, the optimal semi-solid fermentation conditions are: adding 3% calcium carbonate by mass of Astragalus powder and allowing it to ferment at 37℃ for 24 hours.

[0128] Example 4: Preparation of Fermented Astragalus Products

[0129] This embodiment describes the large-scale fermentation of Astragalus membranaceus raw materials (100kg batch) based on the optimal fermentation conditions determined in Example 3. The Astragalus membranaceus raw materials were ground and mixed with a physiological saline suspension of Lactobacillus reuteri GY-18 (OD value 0.5-0.6) at a mass ratio of 1:1. Simultaneously, 3% (by weight of the Astragalus membranaceus raw materials) of calcium carbonate was added. The mixture was then allowed to ferment at 37°C for 24 hours. The fermentation product was then dried at low temperature (≤50°C) and pulverized to obtain fermented Astragalus membranaceus powder with a high astragaloside A content.

[0130] The astragaloside A content in the raw material was 0.56 mg / g, and the astragaloside A content in the fermented astragalus powder produced by the method described in this invention was 0.973 mg / g. It can be seen that the astragaloside A content in the fermented astragalus powder can reach more than 1.7 times that of the raw material, and it can be used as a raw material for preparing astragalus health food in dosage forms such as capsules, tablets, granules, and oral liquids.

[0131] In summary, this invention has obtained a strain of *Lactobacillus reuteri* GY-18 that can increase the content of astragaloside A in fermented *Astragalus membranaceus* products through screening. *Lactobacillus reuteri* GY-18 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 202527926. *Lactobacillus reuteri* GY-18 can secrete acetylxylan esterase, which promotes the conversion of astragaloside saponins to astragaloside A, exhibiting high efficiency and specificity in this conversion and strong conversion ability. This invention achieves a significant increase in astragaloside A content within 24 hours using a semi-solid-state fermentation method, demonstrating excellent practicality.

[0132] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. An application of an acetylxylan esterase, characterized in that, The amino acid sequence of the acetylxylan esterase is shown in SEQ ID NO:1, and the application includes any of the following: (1) Catalyzes the conversion of astragaloside saponins into astragaloside A; (2) Increase the content of astragaloside A in products containing astragalus; (3) Prepare food or health products with high astragaloside A content.

2. The application of biomaterials, characterized in that, The biomaterials are: polynucleotides, expression cassettes, and cells; The polynucleotide encodes an acetylxylan esterase with an amino acid sequence as shown in SEQ ID NO:1; The expression cassette contains the polynucleotide; The cells are cells carrying the polynucleotide or expression cassette, or containing the vector, or capable of expressing acetylxylan esterase as shown in SEQ ID NO:1, or recombinant cells; The application includes any of the following: (1) Catalyzes the conversion of astragaloside saponins into astragaloside A; (2) Increase the content of astragaloside A in products containing astragalus; (3) Prepare food or health products with high astragaloside A content.

3. A type of *Lactobacillus reuteri* GY-18, characterized in that, The *Lactobacillus reutrei* GY-18 expressed acetylxylan esterase with the amino acid sequence shown in SEQ ID NO:1; the *Lactobacillus reutrei* GY-18 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252796, deposit date December 8, 2025, deposit address Wuhan, China, and classification name: *Linosilactobacillus reutrei* GY-18.

4. A microbial inoculant, characterized in that, The microbial agent comprises *Lactobacillus reuteri* GY-18 as described in claim 3.

5. The application of *Lactobacillus reuteri* GY-18 as described in claim 3, or the microbial agent as described in claim 4, characterized in that, The application includes any of the following: (1) Catalyzes the conversion of astragaloside saponins into astragaloside A; (2) Increase the content of astragaloside A in products containing astragalus; (3) Prepare food or health products with high astragaloside A content.

6. A method for catalyzing the conversion of astragaloside saponins to astragaloside A, characterized in that, The method includes: Add acetyl xylan esterase, or Lactobacillus reuteri GY-18 as described in claim 3, or the microbial agent as described in claim 4 to a culture medium or raw material containing astragaloside.

7. The method according to claim 6, characterized in that, The method is as follows: activate the Lactobacillus reuteri GY-18 strain as described in claim 3, and then mix the activated seed liquid with Astragalus membranaceus raw material for semi-solid fermentation.

8. The method according to claim 7, characterized in that, The method includes: (1) Activate Lactobacillus reuteri GY-18 to obtain activated seed liquid; Astragalus raw materials are dried, pulverized, and sterilized to obtain Astragalus powder; (2) Centrifuge the obtained seed liquid to collect the bacterial cells, resuspend them in sterile physiological saline, and mix the resuspended bacterial suspension with Astragalus powder at a mass ratio of 1:1 to form a semi-solid fermentation substrate. (3) The obtained semi-solid fermentation substrate was statically cultured at 37℃ for 18-36h to convert astragaloside into astragaloside A.

9. The method according to claim 8, characterized in that, In step (1), the OD value of the seed solution is 0.8-1.0; In step (2), the bacterial suspension and Astragalus powder are in a mass ratio of 1:1; the OD value of the bacterial suspension is 0.5-0.

6. A pH buffer was also added to the semi-solid fermentation substrate; In step (3), the cells are incubated at 37°C for 24 hours.

10. A food product with high astragaloside A content, characterized in that, The food product is prepared by the method described in any one of claims 6 to 9.

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