Lactobacillus plantarum, preparation and application of lactobacillus plantarum in conversion of soybean isoflavone aglycone

Abstract

The invention discloses lactobacillus plantarum, a preparation and application of the lactobacillus plantarum in conversion of soybean isoflavone aglycone, and belongs to the technical field of microorganisms. The lactobacillus plantarum disclosed by the invention is lactobacillus plantarum WY1, and the preservation number of the lactobacillus plantarum WY1 is CGMCC (China General Microbiological Culture Collection Center) No. 36905. The strain has the capability of transforming soybean isoflavone aglycones, yellow serofluid can be used as a culture medium, the soybean isoflavone aglycones can be transformed into hydroxylated soybean isoflavone aglycones, and the synergistic fermentation effect of the strain and saccharomyces cerevisiae Sc45-32 with the preservation number of CGMCC No.39082 is superior to that of single strain fermentation. The method not only has a great application prospect in hydroxylated soybean isoflavone aglycones, but also can improve resource utilization of wastes rich in soybean isoflavone, such as yellow serofluid, and has great economic value and social benefit.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus plantarum, its preparation, and its application in the transformation of soybean isoflavone aglycones. Background Technology

[0002] Soy isoflavones are an important class of natural bioactive substances found in soybeans and their products. They are mainly divided into glycoside forms (such as daidzein, genistein, and genistein) and aglycone forms (such as daidzein, genistein, and genistein). Among these, the aglycone form has attracted much attention due to its higher bioavailability and physiological activity. For example, soy isoflavone aglycones possess various physiological functions, including antioxidant, anti-inflammatory, osteoporosis prevention, and estrogen level regulation, and have broad application prospects in the development of functional foods, health products, and drugs.

[0003] Currently, isoflavones in natural soybeans mainly exist in the form of glycosides, which are difficult for the human body to absorb and utilize directly. They need to be converted into aglycone forms by intestinal microorganisms or in vitro enzymatic hydrolysis. Traditional methods for converting soybean isoflavone aglycones mainly include chemical acid hydrolysis, enzymatic hydrolysis, and microbial fermentation. Although chemical acid hydrolysis has high conversion efficiency, it is prone to producing byproducts and is environmentally unfriendly; although enzymatic hydrolysis is under mild conditions, the enzyme preparations are expensive and have poor stability, which limits its industrial application.

[0004] Microbial fermentation has become a research hotspot due to its mild conditions, environmental friendliness, and low cost. Currently reported microorganisms used for converting soybean isoflavones mainly include lactic acid bacteria, Bacillus, and yeast. Among them, lactic acid bacteria have been extensively studied due to their safety and probiotic properties, but their conversion efficiency, enzyme activity stability, and adaptability to complex substrates still need improvement. Furthermore, most studies focus on the conversion capacity of single strains, with limited systematic research on multi-strain co-fermentation, especially mixed fermentation of lactic acid bacteria and yeast. There is also a lack of screening and process optimization for highly efficient conversion strains targeting specific bioactive metabolites (such as hydroxylated isoflavones).

[0005] Yellow liquid, a byproduct of tofu production, is rich in protein, carbohydrates, and trace amounts of isoflavones, making it a potential fermentation substrate. However, research on the microbial transformation of isoflavones in yellow liquid is currently insufficient, particularly regarding targeted transformation and functional enhancement using locally sourced fermentation strains.

[0006] Therefore, developing a *Lactobacillus plantarum* strain with high β-glucosidase and hydroxylase activity, adaptable to yellow sap substrate, capable of efficiently converting soybean isoflavone glycosides into aglycones and their hydroxylation products, and constructing a co-fermentation system with yeast, has significant theoretical and practical value. Summary of the Invention

[0007] In view of the problems existing in the prior art, the purpose of this invention is to provide a strain of Lactobacillus plantarum, a preparation thereof, and its application in the conversion of soybean isoflavone aglycones.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A strain of Lactobacillus plantarum, specifically Lactobacillus plantarum WY1, with accession number CGMCC No. 36905.

[0009] The application of the above-mentioned Lactobacillus plantarum in the transformation of soybean isoflavone aglycones.

[0010] Based on the above scheme, soybean isoflavone aglycones are converted into 4',6,7-trihydroxyisoflavones.

[0011] Based on the above scheme, yellow liquid was used as the culture medium, and fermentation was carried out using Lactiplantibacillus plantarum WY1 with preservation number CGMCC No.36905.

[0012] Based on the above scheme, the fermentation conditions are: static fermentation at 37℃ for 18~48 hours.

[0013] Based on the above scheme, the inoculation amount of Lactiplantibacillus plantarum WY1 with accession number CGMCC No.36905 is 0.5-5%.

[0014] Based on the above scheme, the yellow liquid is soybean product yellow liquid.

[0015] Based on the above scheme, the soy product yellow liquid is a liquid byproduct produced during the production of soy products from soybeans after pressing or filtration.

[0016] Based on the above scheme, the soy product is tofu or dried bean curd sticks.

[0017] A formulation for converting soybean isoflavone aglycones, the active ingredients being Lactobacillus plantarum WY1 with preservation number CGMCC No.36905 and Saccharomyces cerevisiae Sc45-32 with preservation number CGMCC No.39082.

[0018] A method for efficiently converting soybean isoflavone aglycones involves inoculating Lactobacillus plantarum WY1 (CGMCC No. 36905) and Saccharomyces cerevisiae Sc45-32 (CGMCC No. 39082) into a yellow slurry medium for fermentation at a ratio of (1:2) to (2:1), with a total inoculum size of 0.5-5%.

[0019] Based on the above scheme, the inoculation ratio of Lactiplantibacillus plantarum WY1 with accession number CGMCC No.36905 and Saccharomyces cerevisiae Sc45-32 with accession number CGMCC No.39082 was 1:2.

[0020] Based on the above scheme, the fermentation conditions are 28~32℃, 140~160 r / min, and fermentation for 18~48 h.

[0021] Based on the above scheme, the yellow liquid is soybean product yellow liquid.

[0022] Based on the above scheme, soybean isoflavone aglycones are converted into 4',6,7-trihydroxyisoflavones.

[0023] Advantages of the technical solution of this invention: This invention isolates a strain of *Lactobacillus plantarum* and a strain of *Saccharomyces cerevisiae* from the sour whey produced during tofu pressing. Both the lactic acid bacteria (D1) and the yeast (D10) possess the ability to convert daidzein (Glycitein, Daidzein, Genistein) into hydroxylated daidzein (4',6,7-Trihydroxyisoflavone) using sour whey as a culture medium. Currently, the hydroxylation conversion of daidzein largely relies on molecular methods. However, both lactic acid bacteria (D1) and yeast (D10) in this invention can hydroxylate daidzein to 4',6,7-Trihydroxyisoflavone through natural fermentation. The conversion method is simple, low-cost, and environmentally friendly. The synergistic fermentation of lactic acid bacteria (D1) and yeast (D10) is more effective than fermentation with a single strain. Therefore, the lactic acid bacteria D1 and yeast D10 of the present invention not only have great application prospects in hydroxylated soybean isoflavone aglycones, but also improve the utilization of waste resources rich in soybean isoflavones, such as yellow pulp water, which has great economic value and social benefits. Attached Figure Description

[0024] Figure 1 Colony morphology and cell morphology of lactic acid bacteria D1; Figure 2 Colony morphology and cell morphology of yeast D10; Figure 3 Phylogenetic tree of lactic acid bacteria D1; Figure 4 Phylogenetic tree of yeast D10; Figure 5 Results of the chromogenic assay for lactic acid bacteria D1 (left image is the negative control, right image is the chromogenic assay result of lactic acid bacteria D1 through puncture). Figure 6 Results of the yeast D10 aescin colorimetric assay (left image is negative control, right image is yeast D10 puncture colorimetric results). Figure 7 Image of the appearance of the yellow liquid culture medium; Figure 8 Determination of the ability of lactic acid bacteria D1 in yellow pulp water to convert isoflavone glycosides; Figure 9 Determination of the ability of yeast D10 in yellow sap to convert isoflavone glycosides; Figure 10 The results are the assay results for the hydroxylase activity of lactic acid bacteria D1; Figure 11 The results are the assay results for hydroxylase activity in yeast D10. Figure 12 Mass spectrum of non-target metabolite 4',6,7-Trihydroxyisoflavone; Figure 13 Extracted ion chromatogram of 4',6,7-trihydroxyisoflavone detected in targeted metabolic analysis (where RT=5.269 is the peak time of the main peak, at which the concentration of the target substance is highest; RT=5.624 is the marking time of a small peak to the right of the main peak and the tail; Q271.00>168.9(+), where Q is the ion pair, 271.00 > 168.90 is the ion transition / mass-charge ratio conversion, 271.00 is the mass of the parent ion, > represents the fragmentation process, 168.90 is the mass of the daughter ions produced after fragmentation, and (+) is the positive ion mode). Figure 14 The content of soybean isoflavone aglycones in fermented yellow water from lactic acid bacteria D1 and yeast D10; Figure 15 The content of 4',6,7-Trihydroxyisoflavone in the yellow slurry water fermented by lactic acid bacteria D1 and yeast D10; Figure 16 The growth curves of lactic acid bacteria D1 and yeast D10 in yellow slurry water are shown. Detailed Implementation

[0025] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0026] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0027] Example 1 Isolation, purification and identification of strains (1) Isolation and purification of the strain: Using 50 mL sterile centrifuge tubes, naturally fermented sour whey (yellow whey produced by pressing tofu, naturally fermented, taken from Laiwu, Jinan, Shandong) was collected and brought back to the laboratory at 4℃. It was immediately diluted with sterile water to a concentration of 10. -3 10 -4 10 -5 10 -6 10 -7 The diluted acid solution was spread onto MRS and YPD agar plates, respectively. Bacteria spread on MRS agar plates were incubated at 37°C for 24 h; bacteria spread on YPD agar plates were incubated at 28°C for 48 h. Suspected colonies were picked based on colony morphology and streaked onto plates for isolation. This process was repeated 2-3 times until pure single colonies were obtained. The purified single colonies were stored in glycerol tubes at -80°C for later use.

[0028] (2) Identification of strains Morphological identification: The colony morphology and cell morphology of the isolated lactic acid bacteria D1 are as follows: Figure 1 As shown, Lactic acid bacteria D1 colonies are small, round, milky white, with a smooth surface, and are easy to pick up. The colony diameter is about 0.5-3 mm. When Gram stained, they all turn blue-purple or purple rod-shaped and are Gram positive.

[0029] The colony morphology and cell morphology of the isolated yeast D10 are as follows: Figure 2 As shown, yeast D10 colonies are round or irregular in shape, with a raised center, a height of about 0.5-2 mm, milky white, and a diameter of about 1-5 mm; the cell morphology is oval or spherical, which is clearly different from that of bacteria.

[0030] Molecular biological identification: Lactic acid bacteria D1 and yeast D10, obtained through screening, were activated to the logarithmic growth phase. RNA was extracted and reverse transcribed into cDNA. Using cDNA as templates, the 16S rDNA sequences of lactic acid bacteria D1 and the 26S rDNA sequences of yeast D10 were amplified by PCR. The obtained sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The 16S rDNA sequences of lactic acid bacteria D1 and the 26S rDNA sequences of yeast D10 were compared for homology using the NCBI BLAST database to obtain homologous strains of lactic acid bacteria D1 and yeast D10. Phylogenetic trees were constructed using strains highly homologous to lactic acid bacteria D1 and yeast D10 to determine their species classification.

[0031] SEQ ID NO:1 (5'→3'): CGAGGCTGAGGCTCCAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGAAAGGGGGGAACCCGAAAATAA SEQ ID NO:2(5’→3’) ACTGACCTGCGGAGGTCATTAAAGAAATTTAATAATTTTGAAAATGGATTTTTTTGTTTTGGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAGTGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTGTTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATTTATTCATTAAATTTTTGTCAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAACATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTTTTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAACTGCGGCTAATCTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCGAACAATGTTCTTAAAGTTTGACCTCAAATCAGGTAGGAGTACCCGCTGAACTTAACATACAAGGGGGGGGGGAGAGAGAGAGAGGGGTTTT The phylogenetic tree of Lactobacillus D1 is as Figure 3As shown, the BLAST database alignment results indicate that lactic acid bacteria D1 shares the highest homology with *Lactiplantibacillus plantarum* strain SK-1, with a sequence similarity of 99.47%. Based on morphological and physiological-biochemical characteristics, strain D1 was identified as *Lactiplantibacillus plantarum*, named *Lactiplantibacillus plantarum* WY1. This strain was deposited on December 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36905. The recommended classification name is *Lactiplantibacillus plantarum*. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] Phylogenetic tree of yeast D10 as follows Figure 4 As shown, the BLAST database alignment results indicate that yeast D10 shares the highest homology with *Saccharomyces cerevisiae* strain HBUAS61417, with a sequence similarity of 99.75%. Based on morphological and physiological-biochemical characteristics, strain D10 was identified as *Saccharomyces cerevisiae*, named *Saccharomyces cerevisiae* Sc45-32. This strain was deposited on December 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 39082. The recommended classification name is *Saccharomyces cerevisiae*. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Example 2 Identification of the hydrolytic ability of lactic acid bacteria D1 and yeast D10 to soybean isoflavone glycosides (1) Cellobiose plate method MRS-cellobiose agar and YPD-cellobiose agar media with cellobiose as the carbon source were prepared: 2% cellobiose was added to sugar-free MRS and YPD agar media as the carbon source, and the media were sterilized at 121℃ for 20 min. Lactic acid bacteria D1 and yeast D10 were streaked onto MRS-cellobiose agar and YPD-cellobiose agar media, respectively, for isolation. The lactic acid bacteria were incubated statically at 37℃ for 24 h, and the yeast was fermented at 28℃ for 24 h. The growth of lactic acid bacteria D1 and yeast D10 on cellobiose agar plates was observed. In cellobiose agar, cellobiose is the only carbon source. β-glucosidase can hydrolyze cellobiose into glucose; therefore, only strains that secrete β-glucosidase can grow on cellobiose agar plates. Both lactic acid bacteria D1 and yeast D10 can grow on cellobiose agar plates, indicating that both lactic acid bacteria D1 and yeast D10 can produce β-glucosidase.

[0034] (2) Screening by aescin colorimetric method The activity of β-glucosidase produced by lactic acid bacteria D1 and yeast D10 was further confirmed by the aescin colorimetric method.

[0035] Preparation of aescin chromogenic medium: Add 0.3% aescin and 0.05% ferric citrate to MRS and YPD agar media, respectively. Initiate the chromogenic reaction of lactic acid bacteria D1 and yeast D10 on the aescin chromogenic medium using the toothpick puncture method. Results are as follows: Figure 5 and Figure 6 As shown, strains secreting β-glucosidase can hydrolyze aescin in the culture medium to produce glucose and esculin. The generated esculin reacts with the iron ions from ferric citrate added beforehand in the culture medium to form a dark brown or black ferrophenolic complex. The larger the black circle and the darker the color, the stronger the enzyme production capacity. Figure 5 and Figure 6 As shown, lactic acid bacteria D1 and yeast D10 have distinct black halos with dark brown edges, indicating that they have a strong ability to produce β-glucosidase.

[0036] (3) β-glucosidase activity assay Microbial fermentation broth: Lactic acid bacteria D1 and yeast D10 were ultrasonically disrupted (ice bath, power 20% or 200 W, ultrasonication for 3 seconds, 10-second intervals, repeated 30 times), centrifuged at 2-8℃ for 20 minutes (5000 r / min), and the supernatant was collected for enzyme activity detection using a microbial β-glucosidase (β-glu) ELISA kit (Jiangsu Enzyme Immunosorbent Assay). The results showed that the β-glucosidase activity of lactic acid bacteria D1 was 56.59±0.80 U / mL, and the β-glucosidase activity of yeast D10 was 44.32±0.95 U / mL.

[0037] Example 3 Nutritional composition determination of yellow slurry after fermentation by lactic acid bacteria D1 and yeast D10 (1) Preparation of soybean milk: Wash 500 g of dried soybeans and soak them in water overnight at room temperature. Then drain and rinse. Take 1000 g of wet soybeans and mix them with 5400 mL of water (the ratio of wet soybeans to water = 1:5.4 w / v) in a soybean milk grinder (model FSM-100, Shenyang Machinery Factory No. 3, China). Filter the resulting slurry through gauze to obtain soybean milk. Heat the soybean milk to 100℃ and maintain for 5 minutes. Take 700 mL of hot soybean milk (100℃) and put it into a stainless steel container, then cool it to 80±2℃. Slowly pour the acidic whey (fermentation) solution (purchased from Li Ji Long Qian Cheng Tofu Shop in Laiwu, Jinan, Shandong) into the steel container while stirring slowly and stopping the addition of acidic whey until the soybean milk coagulates. The volume of acidic whey used is about 185 mL.

[0038] (2) Preparation of yellow slurry culture medium: The fresh yellow slurry prepared in step (1) above is centrifuged at 4000 rpm for 10 min to remove insoluble matter. The supernatant is then sterilized at 121℃ for 15 min to obtain yellow slurry culture medium.

[0039] The appearance of the yellow pulp culture medium is as follows Figure 7 As shown, The moisture, fat, protein, and total sugar contents of three batches of yellow aqueous culture medium prepared by the above method were determined. Moisture content was determined according to GB 5009.3-2016, fat content according to GB 5009.6-2016, protein content according to GB 5009.5-2016, and total sugar content according to GB / T 15672-2009. The results are shown in Table 1.

[0040] Table 1. Nutritional composition analysis of yellow pulp water Note: Different lowercase letters represent significant differences, p<0.05.

[0041] As shown in Table 1, the yellow liquid exhibits a certain degree of batch stability. The yellow liquid contains a relatively high amount of water and a certain amount of carbon and nitrogen sources, which are essential nutrients for microbial growth. It possesses the basic conditions for microbial growth and can be used for microbial fermentation. The test results showed no significant differences, indicating good batch stability of the yellow liquid.

[0042] (3) Yellow slurry culture medium for fermentation of lactic acid bacteria D1 and yeast D10: Take 200 μL of each of the lactic acid bacteria D1 and yeast D10 stored at -80℃ and inoculate them into MRS and YPD media, respectively. The lactic acid bacteria D1 is incubated statically at 37℃ for 24 h, and the yeast D10 is activated overnight at 28℃ and 150 r / min. After activation, both are inoculated into the yellow slurry water medium prepared above at an inoculation rate of 1%. The lactic acid bacteria D1 is incubated statically at 37℃ for 24 h, and the yeast D10 is fermented at 28℃ and 150 r / min for 24 h.

[0043] (4) HPLC analysis of soybean isoflavone content in fermented yellow liquid The fermentation broths obtained from the fermentation of lactic acid bacteria D1 and yeast D10, respectively, and the unfermented yellow slurry culture medium were mixed with 80% chromatographic grade methanol solution at a ratio of 1:1. The mixtures were ultrasonically extracted for 1 h at an ultrasonic power of 200 W and 24 kHz, shaken well, centrifuged at 11000 r / min for 20 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and injected into a 1.5 mL sample vial. The vials were then stored at -20℃ for later use in HPLC analysis.

[0044] HPLC determination of soybean isoflavones: Chromatographic conditions: Chromatographic column: C18 column; Detector: Waters 2695 UV detector; Mobile phase: A: 0.1% (v / v) aqueous acetic acid solution, B: 10.1% (v / v) acetic acid acetonitrile solution; Column temperature: 35℃; Detection wavelength: 260 nm; Injection volume: 10 μL; Analysis time: 60 min. Gradient elution conditions are shown in Table 2. Table 2 Gradient elution program Establishment of standard curves: Weigh 1 mg each of daidzein, daidzein, genistein, genistein, daidzein, and daidzein standards, and dissolve them in chromatographic grade 70% DMSO to prepare 500 μg / mL standard stock solutions. Take appropriate amounts of each standard stock solution and prepare mixed standard solutions with concentrations of 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL, and 10 μg / mL using 70% DMSO. Analyze the mixed standard solutions of each concentration using HPLC, determine the peak area of ​​each component at different concentrations, and plot the standard curves for the six standards using linear regression with the standard concentration (x) of each component against the corresponding peak area (y).

[0045] Calculation of isoflavone content in fermented yellow liquid: The peak areas of each component in the fermented yellow liquid were measured by HPLC and substituted into the standard curves of each standard to obtain the concentration of each component in the sample. The contents of daidzin, gycitin, genistin, daidzein, gycitein, genistein, and the percentage of daidzein aglycones were also calculated. The results of isoflavone content in the fermented yellow liquid by lactic acid bacteria D1 are as follows: Figure 8 As shown, compared with the unfermented control group (CK), the total soybean isoflavone glycosides in the yellow liquid after fermentation by lactic acid bacteria D1 decreased from 301.493±2.59 mg / mL to 72.156±0.63 mg / mL, while the total soybean isoflavone aglycones increased from 22.737±2.32 mg / mL to 206.278±8.51 mg / mL. It can be seen that during the fermentation process, as the amount of soybean isoflavone glycosides decreased, the amount of aglycones gradually increased. Soybean isoflavone glycosides are gradually converted into aglycones during fermentation, and the proportion of soybean isoflavone aglycones also increased from 7% before fermentation to 74.06%. This shows that lactic acid bacteria D1 has the ability to convert soybean isoflavone glycosides into soybean isoflavone aglycones.

[0046] The results of isoflavone content in yeast D10 fermented yellow slurry water are as follows: Figure 9 As shown, compared with the unfermented control group (CK), the total soybean isoflavone glycosides in the yellow slurry water after fermentation by *Saccharomyces cerevisiae* Sc45-32 decreased from 410.633±13.18 mg / mL to 51.2±0.58 mg / mL, while the total soybean isoflavone aglycones increased from 9.818±1.23 mg / mL to 163.784±4.75 mg / mL. This indicates that during fermentation, as the amount of soybean isoflavone glycosides decreased, the amount of aglycones gradually increased. Soybean isoflavone glycosides were gradually converted into aglycones during fermentation, and the proportion of soybean isoflavone aglycones also increased from 2.33% before fermentation to 76.29%. This demonstrates that *Saccharomyces cerevisiae* Sc45-32 has the ability to convert soybean isoflavone glycosides into soybean isoflavone aglycones.

[0047] Example 4 Assay of hydroxylase activity of lactic acid bacteria D1 and yeast D10 200 μL of lactic acid bacteria D1 and yeast D10 stored at -80℃ were inoculated into MRS and YPD liquid media, respectively. Lactic acid bacteria D1 was incubated statically at 37℃ for 24 h, and yeast D10 was activated overnight at 28℃ and shaken at 150 r / min. After activation, both were inoculated into the prepared yellow slurry water medium at an inoculation rate of 1%. Lactic acid bacteria D1 was incubated statically at 37℃ for 24 h, and yeast D10 was fermented at 28℃ and 150 r / min for 24 h.

[0048] The activities of tyrosinase (TyR), cytochrome P450 hydroxylase (CYPH), and flavin monooxygenase (FMO) were determined using the following methods: ①CYP450 enzyme activity assay: The microbial fermentation broth was subjected to ultrasonic disruption of cells (ice bath, power 20% or 200 W, ultrasonic 3 s, 10 s interval, repeated 30 times), centrifuged at 2-8℃ for 20 minutes (5000 rpm), and the supernatant was collected and measured using a microbial cytochrome P450 hydroxylase (CYPH) ELISA kit (Jiangsu Enzyme Immunosorbent Assay).

[0049] ②Tyrosinase activity assay: The microbial fermentation broth was subjected to ultrasonic disruption of cells (ice bath, power 20% or 200W, ultrasonic for 3 seconds, interval 10 seconds, repeated 30 times), centrifuged at 2-8℃ for 20 minutes (5000 rpm), and the supernatant was collected and measured using a microbial tyrosinase (TyR) ELISA kit (Jiangsu Enzyme Immunosorbent Assay).

[0050] ③ Flavin monooxygenase (FMO) activity assay: The microbial fermentation broth was subjected to sonication to disrupt the cells (ice bath, power 20% or 200 W, sonication for 3 s, interval 10 s, repeated 30 times), centrifuged at 2-8℃ for 20 minutes (5000 rpm), and the supernatant was collected and measured using a microbial flavin monooxygenase (FMO) ELISA kit (Jiangsu Enzyme Immunosorbent Assay).

[0051] The enzyme activities of tyrosinase (TyR), cytochrome P450 hydroxylase (CYPH), and flavin monooxygenase (FMO) in lactic acid bacteria D1 and yeast D10 were measured as follows: Figure 10 and Figure 11 As shown, both lactic acid bacteria D1 and yeast D10 have high levels of tyrosinase (TyR), cytochrome P450 hydroxylase (CYPH), and flavin monooxygenase (FMO) activities.

[0052] Example 5 Application of lactic acid bacteria D1 and yeast D10 in the production of 4',6,7-trihydroxyisoflavones from hydroxylated soybean isoflavone aglycones 1. Non-targeted LC-MS / MS mass spectrometry analysis of soybean isoflavones in fermented yellow liquid 200 μL of lactic acid bacteria D1 and yeast D10 stored at -80℃ were inoculated into MRS and YPD liquid media, respectively. Lactic acid bacteria D1 was incubated statically at 37℃ for 24 h, and yeast D10 was activated overnight at 28℃ and shaken at 150 r / min. After activation, both were inoculated into the prepared yellow slurry water medium at an inoculation rate of 1%. Lactic acid bacteria D1 was incubated statically at 37℃ for 24 h, and yeast D10 was fermented at 28℃ and 150 r / min for 24 h.

[0053] Take 400 μL of the fermented sample, centrifuge at 4℃ for 10 min, collect the supernatant and analyze it. The results are as follows: Figure 12 As shown, (4',6,7-Trihydroxyisoflavone) was detected in the fermentation broths of both Lactobacillus brevis D1 and Saccharomyces cerevisiae D10.

[0054] 2. Targeted LC-MS / MS mass spectrometry analysis of soybean isoflavones in fermented yellow liquid 200 μL of lactic acid bacteria D1 and yeast D10, stored at -80℃, were inoculated into MRS and YPD liquid media, respectively. Lactic acid bacteria D1 was incubated statically at 37℃ for 24 h, while yeast D10 was activated overnight at 28℃ with a shaker at 150 r / min. After activation, both were inoculated individually or in combination into the prepared yellow slurry agar medium at a 1% inoculation rate. For individual inoculation: Lactic acid bacteria D1 was incubated statically at 37℃ for 24 h, while yeast D10 was fermented at 28℃ with 150 r / min for 24 h. For combined inoculation: the inoculation ratio of lactic acid bacteria D1 to yeast D10 was 1:1 (0.5%:0.5%), 1:2 (0.33%:0.67%), or 2:1 (0.67%:0.33%), with a total inoculation rate of 1%, and incubated at 30℃ with a shaker at 150 r / min for 24 h.

[0055] After fermentation, 20 mL of fermentation broth sample was freeze-dried for 72 h and concentrated. Then, it was reconstituted with 10 mL of 80% methanol, extracted by sonication for 1 h, centrifuged at 11000 r / min for 20 min, and the precipitate was removed. Then, 20 mL of 80% chromatographic grade methanol was added, sonicated, and centrifuged at 11000 r / min for 20 min to remove the precipitate. The supernatant of the three parallel groups was collected, and the yellow liquid was used as a blank. The samples were stored at -20℃ for targeted LC-MS / MS analysis.

[0056] The extracted ion chromatogram of 4',6,7-trihydroxyisoflavone detected in targeted metabolic analysis is shown below. Figure 13As shown, the content of soybean isoflavone aglycones in the fermented yellow liquid was measured as follows: Figure 14 As shown, the content of 4',6,7-trihydroxyisoflavones in the fermented yellow liquid is as follows: Figure 15 As shown; Depend on Figures 13-15 It was found that 4',6,7-trihydroxyisoflavones were detected in the yellow slurry fermented by both lactic acid bacteria D1 and yeast D10. After fermentation with Saccharomyces cerevisiae Sc45-32 for 24 h, the total soybean isoflavone aglycone (Glycitein, Daidzein, Genistein) content increased from 403.86±13.80 ng / mL in the unfermented yellow slurry to 11209.74±193.80 ng / mL; the 4',6,7-trihydroxyisoflavone content increased from 6.61±0.20 ng / mL in the unfermented yellow slurry to 403.12±9.06 ng / mL.

[0057] The contents of total soybean isoflavones (Glycitein, Daidzein, Genistein) and hydroxylated soybean isoflavones (4',6,7-Trihydroxyisoflavone) were both highest when lactic acid bacteria D1 and yeast D10 were inoculated at a ratio of 1:2 to ferment yellow slurry. The content of total soybean isoflavones increased from 403.86±13.80 ng / mL in unfermented yellow slurry to 13286.87±785.51 ng / mL. When lactic acid bacteria D1 and yeast D10 were inoculated into fermented yellow slurry at a 1:2 ratio, the content of 4',6,7-Trihydroxyisoflavone increased from 6.61±0.20 ng / mL in the unfermented yellow slurry to 435.39±1.49 ng / mL. When lactic acid bacteria D1 and yeast D10 were inoculated into fermented yellow slurry at 1:1 and 2:1 ratios, the content of 4',6,7-Trihydroxyisoflavone was 383.16±6.95 ng / mL and 311.26±26.04 ng / mL, respectively. After fermentation of lactic acid bacteria D1 alone for 24 hours, the content of 4',6,7-Trihydroxyisoflavone was the lowest at 198.65±13.30 ng / mL.

[0058] 3. Growth curves of lactic acid bacteria D1 and yeast D10 After second-generation passage of lactic acid bacteria D1 and yeast D10 stored at -80℃, they were added to yellow slurry agar medium at a 1% inoculum size. Lactic acid bacteria D1 was cultured in a constant temperature incubator at 37℃ for 24 h, and yeast D10 was cultured in a shaker at 28-30℃ and 150 r / min for 24 h. The OD values ​​after 24 h of fermentation were measured using a microbial growth curve analyzer. 600The OD600 value, or absorbance at 600 nanometers, is a standard indicator for measuring bacterial concentration in microbiological experiments. A higher OD600 value indicates greater turbidity and a higher concentration of bacteria in the bacterial solution. Measurements were taken every 2 hours, using uninoculated yellow liquid as a control. Results are as follows... Figure 16 As shown, lactic acid bacteria D1 and yeast D10 reach the logarithmic growth phase in about 8 hours of fermentation and the plateau phase in about 18 hours. This can be used as the "gold standard" for judging whether the fermentation process is normal, and whether the batch of yellow slurry is stable and contaminated.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus plantarum*, characterized in that, The Lactobacillus plantarum mentioned is Lactiplantibacillus plantarum WY1, with accession number CGMCC No. 36905.

2. The use of the *Lactobacillus plantarum* as described in claim 1 in the transformation of soybean isoflavone aglycones.

3. The application according to claim 2, characterized in that, Soy isoflavone aglycones were converted into 4',6,7-trihydroxyisoflavones.

4. The application according to claim 3, characterized in that, Using yellow liquid as the culture medium, fermentation was carried out using Lactiplantibacillus plantarum WY1 with preservation number CGMCCNo.36905.

5. The application according to claim 4, characterized in that, The yellow liquid is soybean product yellow liquid.

6. A formulation for converting soybean isoflavone aglycones, characterized in that, The active ingredients are Lactobacillus plantarum WY1 with preservation number CGMCC No. 36905 and Saccharomyces cerevisiae Sc45-32 with preservation number CGMCC No. 39082.

7. A method for efficiently converting soybean isoflavone aglycones, characterized in that, Lactobacillus plantarum WY1 (CGMCC No. 36905) and Saccharomyces cerevisiae Sc45-32 (CGMCC No. 39082) were inoculated into yellow slurry medium at a ratio of (1:2) to (2:1), with a total inoculum of 0.5-5%.

8. The method for efficiently converting soybean isoflavone aglycones according to claim 7, characterized in that, The inoculation ratio of Lactiplantibacillus plantarum WY1 (CGMCC No. 36905) and Saccharomyces cerevisiae Sc45-32 (CGMCC No. 39082) was 1:

2.

9. The method for efficiently converting soybean isoflavone aglycones according to claim 8, characterized in that, The fermentation conditions were 30℃, 150 r / min, and 24 h.

10. The method for efficiently converting soybean isoflavone aglycones according to any one of claims 7 to 9, characterized in that, The yellow liquid is soybean product yellow liquid.

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