A method for increasing the proportion of genistein in soybean isoflavone glycosides by directional fermentation

Abstract

The application discloses a method for improving the proportion of genistein in soybean isoflavone glycosides through directional fermentation. Lactobacillus sp. The method is characterized in that Lactobacillus (RS-03027) is used for fermentation after induced pre-culture, and the substrate is fed in batches, so that high selective conversion of isoflavone glycosides to genistein is realized. The strain has high beta-glucosidase activity related conversion capacity, has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.21302. The process realizes high accumulation and high selective enrichment of the target product, effectively inhibits non-target aglycone, and has industrial amplification application potential.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation and directed biotransformation of natural products, specifically to a method for increasing the proportion of genistein in soybean isoflavone glycosides through directed fermentation. Background Technology

[0002] Soy isoflavones mainly exist in two forms: glycosides and aglycones. Glycoside isoflavones include genistein, daidzein, and genistein; aglycone isoflavones include genistein, daidzein, and genistein. Compared to glycosides, aglycones generally have better absorption and utilization characteristics. Therefore, using lactic acid bacteria to convert glycoside isoflavones into aglycone isoflavones has become an important direction in the development of functional soy products.

[0003] In existing technologies, there are many schemes for using lactic acid bacteria in the fermentation of soy milk or soybean systems to increase the content of isoflavone aglycones. However, most schemes mainly focus on increasing the total aglycone content, with insufficient control over specific target aglycones. At the same time, different strains have significant differences in their hydrolytic ability of isoflavone glycosides, and different fermentation conditions can affect the conversion pathway, final product composition, and by-product formation.

[0004] Therefore, although existing technologies can achieve the overall conversion of soybean isoflavone glycosides into aglycone-type isoflavones, there is still room for improvement in increasing the proportion of specific target aglycones, controlling the proportion of non-target aglycones, and enhancing the controllability of the fermentation process. Summary of the Invention

[0005] The purpose of this invention is to provide a method for increasing the proportion of genistein in soybean isoflavone glycosides through directional fermentation. This method addresses the problems in existing technologies, such as insufficient selectivity of target products, high proportion of non-target aglycones, and poor stability of the fermentation process, by inducing pre-cultured strains, feeding in stages, and pH control.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A strain of Lactobacillus exhibiting high selectivity for genistein, the strain being taxonomically named Lactobacillus ( Lactobacillus sp. RS-03027 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 21302, on December 4, 2020.

[0007] This strain exhibits good induction response and transformation adaptability to soybean isoflavone glycoside substrates. It expresses high β-glucosidase activity and has a strict substrate recognition domain. It has a lower affinity for genistein and higher catalytic efficiency, but significantly lower catalytic activity for daidzein.

[0008] Lactobacillus ( Lactobacillus After being cultured on MRS solid medium at 37°C for 24 h, sp.) RS-03027 colonies are generally milky white or white, round, with smooth surface, neat edges, and slightly raised. Under a microscope, the bacteria are generally short rods or rods, mostly arranged singly, in pairs or short chains, Gram-positive, and do not form spores.

[0009] The application of the strain in the preparation of genistein.

[0010] Application of the strain in the targeted conversion and processing of soy milk, soy milk, soy meal extract or soy isoflavone extract.

[0011] The application of the strain in increasing the proportion of genistein in soybean isoflavone glycosides through fermentation.

[0012] The soybean isoflavone glycosides include one or more of genistein, daidzein, and genistein.

[0013] A method for increasing the proportion of genistein in soybean isoflavone glycosides by directed fermentation using strain RS-03027 includes the following steps: (1) Lactobacillus ( Lactobacillus sp. RS-03027 was used for activation culture to obtain activated seed culture; (2) The activated seed liquid was inoculated into an induction pre-culture medium containing inducing components for induction pre-culture to obtain the bacterial liquid after induction pre-culture; The induced pre-cultured bacterial solution was inoculated into the transformation medium, and then soybean isoflavone glycosides were added to the transformation system for fermentation to obtain a transformation system with an increased proportion of genistein.

[0014] The inducing component includes a low dose of soybean isoflavone glycosides and may further contain a fermentable carbon source.

[0015] The induction pre-culture conditions are as follows: inoculum size 2%–10% (v / v), culture temperature 30–37℃, and culture time 6–18 h.

[0016] The conversion medium mentioned in step (3) is: glucose 10-15 g / L, peptone 5-10 g / L, yeast extract 3-8 g / L, K2HPO4 1-2 g / L, KH2PO4 1-2 g / L, MgSO4 0.1-0.5 g / L.

[0017] The total amount of soybean isoflavone glycosides added to the conversion fermentation system is 0.10–1.00 g / L, preferably 0.50 g / L, and the initial pH is 6.2–6.8.

[0018] The soybean isoflavone glycosides are added in 2 to 6 batches, with the first batch accounting for 20% to 50% of the total amount.

[0019] The soybean isoflavone glycosides include one or more of genistein, daidzein, and genistein.

[0020] In this invention, soybean isoflavone glycosides can also be replaced with conversion substrates such as soy milk, soy milk, soybean meal extract, or soybean extract rich in isoflavone glycosides.

[0021] The conversion substrate is added in 2 to 6 stages, with the initial addition being 20% ​​to 50% of the total amount. The total addition of conversion substrate is 0.10 to 1.00 g / L.

[0022] The method of this invention increases the conversion ratio of soybean isoflavone glycosides to genistein, reduces the accumulation of major non-target aglycones represented by daidzein, and increases the proportion of genistein in the total aglycones.

[0023] The method is applied in the targeted conversion and processing of soy milk, soybean meal extract, or soy isoflavone extract.

[0024] The method increases the conversion rate of soybean isoflavone glycosides to genistein, reduces the accumulation of major non-target aglycones represented by daidzein, and thus increases the proportion of genistein in the total aglycones.

[0025] Fermentation is stopped after reaching a preferred termination time point to obtain a conversion system enriched with genistein. The preferred termination time point is determined by one or more of the following indicators: a. the residual genistein content decreases to a stable level; b. the genistein content reaches a peak or enters a stable plateau; c. the proportion of genistein to total aglycones reaches a peak or enters a stable plateau. Preferably, under the conditions of this invention, the preferred termination time point is 24 hours.

[0026] A method for increasing the proportion of genistein in soybean isoflavone glycosides through targeted fermentation, the method using Lactobacillus ( Lactobacillus sp. RS-03027 was fermented in a conversion medium supplemented with soybean isoflavone glycosides to selectively convert the substrate into genistein, thereby increasing the proportion of specific target aglycones.

[0027] The present invention has found that strain RS-03027 has a good induction response and transformation adaptability to soybean isoflavone glycoside substrates, and under the process conditions of the present invention, it is beneficial to the generation of genistein and the increase of its proportion.

[0028] Without departing from the concept of this invention, other lactobacilli with similar glycoside hydrolysis-related transformation capabilities can be adapted and optimized by referring to the process of this invention.

[0029] This invention further combines induction pre-culture, multi-stage feeding, pH control, and termination time point control to construct a directional transformation process that matches the strain, thereby achieving highly selective conversion of soybean isoflavone glycosides to genistein, increasing the production of the target product genistein and its proportion in the main aglycone products, and reducing the accumulation of the main non-target aglycones, represented by daidzein.

[0030] (1) Induction pre-culture: The cells were pre-induced in an induction medium containing low doses of isoflavone glycosides to enhance the metabolic response related to glycoside hydrolysis in the early stage of fermentation.

[0031] (2) Staged feeding: Isoflavone glycosides are added in stages to reduce the instantaneous substrate concentration, alleviate substrate / product inhibition and maintain continuous conversion.

[0032] (3) pH control: Maintain a suitable pH range during fermentation to provide a stable environment for glycoside hydrolysis and directional conversion.

[0033] (4) Termination time control: Fermentation is terminated when the proportion of the target product reaches its peak or enters the stable period to avoid further accumulation of non-target aglycones.

[0034] Through the above combined control, high accumulation and high enrichment of the target product can be achieved, and this can be extended to systems such as soy milk, soy milk, and soy meal extract.

[0035] Revive the preserved Lactobacillus RS-03027 from the preservation tube or cryopreservation tube, inoculate it into the basal culture medium, and culture it at 30-37℃ for 6-24 hours to obtain activated liquid seed culture medium.

[0036] The basal culture medium is MRS liquid culture medium or its modified culture medium.

[0037] Inoculate the activated seed liquid culture medium into the induction pre-culture medium at an inoculation rate of 2% to 10% (v / v) and culture at 30 to 37°C for 6 to 18 hours to obtain the induction pre-cultured bacterial solution.

[0038] The inducing component includes a low dose of soybean isoflavone glycosides and / or a fermentable carbon source. "Low dose" refers to a relatively low level of addition during the induction pre-culture stage relative to the total amount of soybean isoflavone glycosides added during the conversion fermentation stage. The soybean isoflavone glycoside content in the inducing component is 0.01–0.20 g / L.

[0039] Preferably, the fermentable carbon source is one or more of glucose, maltose, and lactose, with glucose being the most preferred, and the amount added is 5-15 g / L.

[0040] The inducing component is used to enhance the response level of the cell-associated glycoside hydrolysis pathway.

[0041] The induction pre-culture medium consists of: glucose 20–25 g / L, peptone 10–15 g / L, beef extract 10–15 g / L, yeast extract 3–8 g / L, ammonium citrate 2–3 g / L, sodium acetate 4–6 g / L, dipotassium hydrogen phosphate 1–2 g / L, MgSO4·7H2O 0.1–0.5 g / L, MnSO4·H2O 0.05–0.1 g / L, Tween-80 1 mL / L, and soybean isoflavone glycosides 0.01–0.20 g / L. In addition, other fermentable carbon sources may be added at 5–15 g / L.

[0042] In the process of this invention, a low dose of soybean isoflavone glycosides is used for induction, and a high dose is used for the accumulation of the target product. The low dose is 0.01–0.20 g / L, and the high dose is 0.10–1.00 g / L.

[0043] The induction pre-culture medium can be appropriately replaced by soy milk, soy milk, soy meal extract or soybean extract rich in isoflavone glycosides to obtain the induction pre-culture effect, and is not limited to the above formula.

[0044] During fermentation, the temperature is controlled at 28–37℃, the pH at 4.0–6.0, and the time at 6–48 h. The mixture is allowed to stand and is kept under low or micro-aerobic conditions.

[0045] Preferably, fermentation is terminated when the genistein content reaches its peak and the non-target aglycone does not increase significantly.

[0046] After fermentation is terminated, the bacterial cells are isolated or the fermentation broth is directly taken and analyzed by HPLC or UPLC to detect the following: genistein residue, genistein content, daidzein and daidzein content, and the proportion of genistein to total aglycones.

[0047] Dissolve 0.5 mM pNPG in 500 μL of 5 mM phosphate / citric acid buffer (pH 6.8), add 500 μL of the prepared bacterial suspension, and react at 40℃ for 30 min. After the reaction, add 500 μL of 2 M Na2CO3 to terminate the reaction. Centrifuge at 12000–14000 rpm for 15 min, and measure the absorbance of the supernatant at 400 nm. 1 U is defined as the amount of enzyme that releases 1 μmol of p-nitrophenol per minute per mL of reaction system.

[0048] HPLC chromatographic detection can be performed under the following conditions: Mobile phase A: water:methanol:glacial acetic acid = 88:10:2; mobile phase B: methanol solution containing 2% glacial acetic acid; flow rate: 0.4 mL / min; detection wavelength: 260 nm; column temperature: 32℃; injection volume: 20 μL. Standard solutions were prepared using genistein, genistein flavonoids, daidzein, daidzein, daidzein glycosides, and daidzein standards to establish standard curves. Sample filtrates were analyzed, and the content of each component in the sample was calculated based on the standard curves. The proportion of genistein flavonoids in the total aglycones was calculated, and the residual amount of genistein and the proportion of non-target aglycones were statistically analyzed.

[0049] "Total aglycones" refers to the main aglycone products detected in this invention, namely the sum of genistein and daidzein. Genistein percentage (%) = genistein / (genistein + daidzein) × 100%.

[0050] The technical solution of this invention not only relies on unique Lactobacillus sp.RS-03027 The study highlights the high activity of β-glucosidase and proposes a clear combined operation strategy to achieve overall control over the conversion process of glycosides to aglycones, resulting in a significantly higher accumulation of the target product, genistein, and its proportion in the total aglycones compared to existing conventional techniques.

[0051] Beneficial effects:

[0052] The lactobacillus of the present invention RS-03027 This invention exhibits high selectivity for genistein and significantly promotes the increase of β-glucosidase activity in the early stages of fermentation during the conversion of soybean isoflavone glycosides, thereby improving the conversion efficiency of soybean isoflavone glycosides to the target product, genistein. At the 24-hour fermentation endpoint, the cumulative level of genistein was significantly higher than that of the control group, indicating that this invention can achieve a higher level of target product formation.

[0053] This method not only increases the total amount of the target product but also increases the proportion of the target product in the total aglycones. Compared with conventional methods, it is more conducive to achieving selective enrichment of the target product and improves the composition and structure of the final product. The accumulation level of non-target products in the fermentation system is controlled within a low range, thereby reducing the difficulty of product separation and improving product purity.

[0054] It is beneficial for industrial application and process stability, and can provide technical support for large-scale implementation in industrial production processes, enabling stable and efficient production. Attached Figure Description

[0055] Figure 1 Effects of induced pre-culture on changes in β-glucosidase activity during the early stage of fermentation; Figure 2 Genistein formation curves for each example; Figure 3: Curves showing changes in daidzein in each example; Figure 4 Curves showing the change in the proportion of genistein over time in each embodiment; Figure 5 Comparison of the main aglycone concentrations in each example at 24 hours; Figure 6 Comparison of the proportion of genistein in each embodiment at 24h, the proportion is calculated as genistein / (genistein + daidzein) × 100%. Detailed Implementation

[0056] The present invention can be better understood through the following embodiments. Those skilled in the art will readily understand that the specific process conditions and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0057] The strains involved in the following examples are: the starting strain Lactobacillus ( Lactobacillus sp. CGMCC No. 21302, originating from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 21302.

[0058] The standard substances genistein, genistein flavonoids, daidzein, daidzein, daidzein, daidzein and daidzein mentioned in the following examples can all be purchased from commercial standard suppliers.

[0059] In the following examples, methanol and acetonitrile were HPLC grade, and glacial acetic acid, NaOH, and phosphate buffer were analytical grade reagents.

[0060] The strains used in the following examples are strains Lactobacillus sp. RS-03027, this strain was screened from soil samples of the green belt on the campus of Nanjing University of Technology. Soil samples were added to sterile physiological saline and shaken to mix. The supernatant was then serially diluted and plated onto MRS solid agar plates and incubated at 37℃ for 24–48 h. Single colonies were picked for isolation and purification, and stable strains were obtained through continuous subculturing. After 24 h of incubation on MRS solid agar at 37℃, the colonies were milky white or white, round, with a smooth surface, regular edges, and slightly raised. Under a microscope, the bacteria were generally short rods or rod-shaped, mostly arranged singly, in pairs, or in short chains. They were Gram-positive, did not form spores, and were identified as *Lactobacillus* (Lactobacillus). Lactobacillus sp.), named Lactobacillus ( Lactobacillussp.) RS-03027, and was deposited on December 4, 2020 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.21302.

[0061] Example 1: Induction Pre-culture and Fertilization MRS liquid culture medium: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, K2HPO4 2 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·H2O 0.05 g / L, Tween-80 1 mL / L, adjust the initial pH to 6.2–6.8, and sterilize at 121℃ for 15 min.

[0062] MRS solid medium: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, K2HPO4 2 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·H2O 0.05 g / L, Tween-80 1 mL / L, agar 15–20 g / L, adjust the initial pH to 6.2–6.8, sterilize at 121℃ for 15 min.

[0063] Induction pre-medium: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, K2HPO4 2 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·H2O 0.05 g / L, Tween-80 1 mL / L, soybean isoflavone glycosides 0.1 g / L, maltose 5 g / L. Adjust the initial pH to 6.2–6.8 and sterilize at 121℃ for 15 min.

[0064] Transformation medium: glucose 15 g / L, peptone 10 g / L, yeast extract 5 g / L, K2HPO4 1.5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.2 g / L, adjust the initial pH to 6.5, and sterilize at 121℃ for 15 min.

[0065] (1) Take the lactobacillus out of the cryopreservation tube, streak it onto an MRS solid medium plate, and incubate it at 37°C for 24 hours.

[0066] (2) Pick a single colony from the solid culture medium and inoculate it into 50 mL of MRS liquid culture medium. Incubate at 37℃ and 180 rpm for 16 h to obtain a primary activated seed culture.

[0067] (3) Take the above-mentioned primary activated seed solution and transfer it to a new MRS liquid medium at an inoculation rate of 5% (v / v). Continue to culture at 37℃ and 180 rpm for 12 hours to obtain the secondary activated seed solution.

[0068] (4) Take the secondary activated seed liquid and measure the OD600 of the bacterial culture. Then perform serial dilution and spread it on plates to determine the viable count (CFU / mL). The viable count is determined by plate count method. The result is expressed as CFU / mL. The viable count is used to monitor the stability and repeatability of the fermentation process and is not used as the core indicator for the selective evaluation of directional transformation in this invention.

[0069] (5) The above secondary seed culture was inoculated into the induction pre-culture medium at an inoculation rate of 5% (v / v) and cultured at 35℃ and 180 rpm for 12 h to obtain the bacterial culture after induction pre-culture.

[0070] After the above culture was completed, a portion of the bacterial suspension was used to determine the OD600. Another portion of the bacterial suspension was serially diluted and spread onto MRS solid medium plates. After incubation at 37°C for 24 hours, the viable cells were counted to determine the number of viable cells after induction pre-culture. The induction pre-cultured bacterial suspension was centrifuged (8000–10000 rpm, 10 min), the supernatant was discarded, and the cells were resuspended in sterile PBS to adjust the OD600 of the bacterial suspension to 1.0 for later use in determining β-glucosidase activity. Sterilized transformation medium was taken and cooled to room temperature for later use.

[0071] The inducing pre-cultured bacterial suspension was inoculated into the transformation medium at a 5% (v / v) inoculation rate. Based on a total soybean isoflavone glycoside addition of 0.50 g / L, the total addition amounts of genistein, daidzein, and genistein were 0.25 g / L, 0.20 g / L, and 0.05 g / L, respectively, added to the transformation medium in multiple additions. The first addition was 50% of the total substrate, and the remaining 50% was added in five equal increments, each time adding 10% of the total substrate, every 1 hour. The inoculated Erlenmeyer flasks containing the bacterial suspension were placed in a shaker and fermented at 37℃ and 180 rpm. During fermentation, samples were taken every 2–4 hours to check the pH value; simultaneously, the pH of the system was adjusted to 4.5–5.5 to maintain the overall fermentation process within the pH range of 4.0–6.0. ​​Sampling time points were: 0h, 2h, 4h, 6h, 8h, 12h, 18h, and 24h. Take 2–5 mL samples at each time point and immediately place them on ice. Terminate fermentation after 24 h. Centrifuge the samples from each time point at 12000 rpm for 10 min to separate the cells and supernatant. Accurately weigh 10 g of the sample to be tested and add methanol:water = 4:1 (v / v) extraction buffer to a final volume of 80 mL. Extract the sample in a 65℃ water bath for 4 h. After extraction, cool to room temperature, add 6 mL of 2 mol / L NaOH, shake at 200 rpm for 10 min, and then add 4 mL of glacial acetic acid to neutralize. Make up the volume and mix well. Centrifuge 20 mL of the sample solution at 12000 rpm for 10 min. Filter the supernatant through a 0.45 μm organic filter membrane and collect the filtrate for HPLC analysis.

[0072] Example 2

[0073] The culture medium formulation in this embodiment is the same as that in Example 1, except that it was not induced by the pre-culture medium in step 5), and the total amount of soybean isoflavone glycosides 0.50 g / L was added to the transformation medium all at once, without being added in batches.

[0074] The plate resuscitation, primary activation, secondary activation, and viable cell count determination methods for the strains are the same as in Example 1, and the β-glucosidase activity determination method is the same as in Example 1.

[0075] The secondary activated seed liquid obtained in step 4) of Example 1 was used as the fermentation inoculum and inoculated into the transformation medium at a 5% (v / v) inoculation rate. Based on a total soybean isoflavone glycoside addition of 0.50 g / L, the addition amounts of genistein, daidzein, and genistein were 0.25 g / L, 0.20 g / L, and 0.05 g / L, respectively, and were added to the transformation medium all at once during inoculation. The inoculated Erlenmeyer flasks were placed in a shaker and fermented at 37°C and 180 rpm. During fermentation, samples were taken every 2–4 hours to check the pH value, and the pH of the system was adjusted to 4.5–5.5 to maintain the overall fermentation process within the pH range of 4.0–6.0. ​​Sampling time points were: 0h, 2h, 4h, 6h, 8h, 12h, 18h, and 24h. 2–5 mL samples were taken at each time point and immediately placed on ice for later use. Under the conditions of this embodiment, the endpoint comparison was performed with 24 hours as the termination point, and the β-glucosidase activity was determined according to the method described in Example 1.

[0076] Example 3

[0077] The culture medium formulation in this embodiment is the same as that in Example 1, except that the total amount of soybean isoflavone glycosides 0.50 g / L was added to the transformation medium all at once at the time of inoculation, and was not added in batches.

[0078] The starting strain of Lactobacillus ( ) was prepared according to the method in Example 1. Lactobacillus sp. The plate resuscitation, primary activation, secondary activation, induction pre-culture, and β-glucosidase activity assay of RS-03027 were performed. The inducing pre-cultured bacterial culture was inoculated into transformation medium at a 5% (v / v) inoculation rate. Based on a total soybean isoflavone glycoside addition of 0.50 g / L, the addition amounts of genistein, daidzein, and genistein were 0.25 g / L, 0.20 g / L, and 0.05 g / L, respectively, and were added to the transformation medium all at once during inoculation. The inoculated Erlenmeyer flasks were placed in a shaker and fermented at 37℃ and 180 rpm. During fermentation, samples were taken every 2–4 hours to check the pH value; simultaneously, the pH of the system was adjusted to 4.5–5.5 to maintain the overall fermentation process within the pH range of 4.0–6.0. ​​Sampling time points were: 0h, 2h, 4h, 6h, 8h, 12h, 18h, and 24h. 2–5 mL samples were taken at each time point and immediately placed on ice for later use. Under the conditions of this embodiment, fermentation was terminated after 24 hours.

[0079] Example 4

[0080] The culture medium formulation in this embodiment is the same as that in Example 1, except that step 5) of induction pre-culture was not performed. The starting strain Lactobacillus (…) was cultured according to steps 1) to 4) in Example 1. Lactobacillus sp.The plate resuscitation, primary activation, secondary activation, and viable count determination of RS-03027 were performed. The secondary activated seed culture obtained in step 4) of Example 1 was used as the fermentation inoculum. The secondary activated seed culture was inoculated into the transformation medium at a 5% (v / v) inoculation rate. Based on a total soybean isoflavone glycoside addition of 0.50 g / L, the total addition amounts of genistein, daidzein, and genistein were 0.25 g / L, 0.20 g / L, and 0.05 g / L, respectively, and were added to the transformation medium in multiple additions. The first addition was 50% of the total substrate, and the remaining 50% was added in five equal increments, each time adding 10% of the total substrate, every 1 hour. The inoculated Erlenmeyer flasks were placed in a shaker and fermented at 37°C and 180 rpm. During fermentation, samples were taken every 2–4 hours to check the pH value; simultaneously, the pH of the system was adjusted to 4.5–5.5, ensuring the overall fermentation process remained within the pH range of 4.0–6.0. Sampling times were: 0h, 2h, 4h, 6h, 8h, 12h, 18h, and 24h. 2–5 mL samples were taken at each time point and immediately placed on ice for later use. Under the conditions of this example, fermentation was terminated after 24h, and the β-glucosidase activity assay was performed using the same method as in Example 2.

[0081] Example Results and Analysis: The present invention compares Examples 1 to 4 under the same detection method. Figure 1 This reflects the changes in β-glucosidase activity during the early stage of fermentation in Examples 1-4; Figure 2 , Figure 3 The dynamic changes of the target product genistein and the main non-target aglycone daidzein in Examples 1-4 during the 0-24h process are reflected respectively. Figure 4 The changes in the proportion of genistein in Examples 1-4 over time; Figure 5 This is a summary comparison of the endpoint concentrations of major aglycone at 24 h for Examples 1-4. Figure 6 This is a summary comparison of the percentage of genistein in Examples 1-4 at 24 hours.

[0082] (1) Effect of induced pre-culture on β-glucosidase activity in the early stage of fermentation: such as Figure 1 As shown, the β-glucosidase activity in Examples 1 and 3 (including induced pre-culture) was increased from 0 to 6 h. Compared with Examples 2 and 4 (without induced pre-culture), this indicates that induced pre-culture can increase the activity levels of glycoside hydrolysis-related enzymes in the early stage of fermentation and accelerate the conversion initiation. Taking 6 h as an example, the β-glucosidase activity in Example 1 was 1.24 U / mL, and in Example 2 it was 0.64 U / mL, an increase of 93.75%.

[0083] (2) Formation curve of the target product, genistein: as shown Figure 2As shown, Example 1 showed the fastest genistein production rate and the highest cumulative endpoint, Example 2 showed the lowest overall rate, and Examples 3 and 4 were in between. This indicates that the combination of induced pre-culture and fractional feeding is beneficial for the continuous generation of the target product and improves the endpoint level.

[0084] (3) Major non-target aglycone changes: such as Figure 3 As shown, under the conditions of this invention, genistein is one of the main forms of non-target aglycones, so genistein is used as a representative indicator of the main non-target aglycones for analysis. The results show that the cumulative level of genistein in Example 1 is significantly lower than that in Examples 2 and 3, indicating that the process of this invention has an inhibitory effect on non-target pathways; the highest accumulation of genistein was observed in Example 3 (only induction pre-culture), indicating that improving the transformation initiation ability may be accompanied by an increase in non-target aglycones.

[0085] (4) Changes in the proportion of flavonoids in genistein over time: e.g. Figure 4 As shown, the proportion of genistein in each group increased rapidly in the early stage of fermentation and then tended to stabilize. Among them, the proportion in Example 1 was the highest overall and remained stable in the later stage, while the proportion in Example 2 was the lowest. This indicates that the process of the present invention can increase the absolute amount of the target product while increasing its relative proportion in the total aglycone.

[0086] (5) Comparison of major aglycone concentrations over 24 hours: e.g. Figure 5 As shown, at 24h, the concentration of genistein in Example 1 was 146.20 mg / L, while that in Example 2 was 73.80 mg / L, an increase of 98.10%; the concentration of daidzein in Example 1 was 34.50 mg / L, while that in Example 2 was 85.40 mg / L, a decrease of 59.60%.

[0087] (6) Comparison of the proportion of genistein to total aglycones in 24-hour genistein: e.g. Figure 6 As shown, at 24 hours, the proportion of genistein in the total aglycones in Example 1 was 77.52%, while in Example 2 it was 44.56%, a relative increase of 74.01%. In Example 4, it was 65.59%, higher than the 46.81% in Example 3, indicating that multiple feedings have a promoting effect on increasing the proportion, and the combination of induction pre-culture and multiple feedings (Example 1) can further achieve the highest level of proportion.

[0088] (7) In this embodiment of the invention, the target product ratio reaches a stable plateau at 24 h and the non-target aglycones do not accumulate further, so it is used as a preferred termination time point for endpoint comparison.

Claims

1. A strain exhibiting high selectivity for genistein, characterized in that, The strain was taxonomically named Lactobacillus (Lactobacillus) Lactobacillus sp. RS-03027 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21302.

2. The use of the strain described in claim 1 in the preparation of genistein.

3. The application of the strain described in claim 1 in increasing the proportion of genistein in soybean isoflavone glycosides through fermentation.

4. A method for increasing the proportion of genistein in a conversion substrate through targeted fermentation, characterized in that, Includes the following steps: (1) Lactobacillus ( Lactobacillus sp. RS-03027 was used for activation culture to obtain activated seed culture; (2) The activated seed liquid was inoculated into an induction pre-culture medium containing inducing components for induction pre-culture to obtain the bacterial liquid after induction pre-culture; (3) The bacterial culture after induction and pre-culture was inoculated into the transformation medium, and then the transformation substrate was added to the transformation system for fermentation to obtain a transformation system with an increased proportion of genistein.

5. The method according to claim 4, characterized in that, The induction pre-culture medium mentioned in step (2) consists of 20-25 g / L glucose, 10-15 g / L peptone, 10-15 g / L beef extract, 3-8 g / L yeast extract, 2-3 g / L ammonium citrate, 4-6 g / L sodium acetate, 1-2 g / L dipotassium hydrogen phosphate, 0.1-0.5 g / L MgSO4·7H2O, 0.05-0.1 g / L MnSO4·H2O, 1 mL / L Tween-80, and 0.01-0.20 g / L soybean isoflavone glycosides.

6. The method according to claim 4, characterized in that, The conversion medium mentioned in step (3) is: glucose 10-15 g / L, peptone 5-10 g / L, yeast extract 3-8 g / L, K2HPO4 1-2 g / L, KH2PO4 1-2 g / L, MgSO4 0.1-0.5 g / L.

7. The method according to claim 4, characterized in that, The conversion substrate mentioned in step (3) is any one of soy milk, soy milk, soybean meal extract, or soybean extract rich in isoflavone glycosides and soybean isoflavone glycosides.

8. The method according to claim 4, characterized in that, The conversion substrate is added in 2 to 6 batches, with the initial batch amount being 20% ​​to 50% of the total batch amount, and the total addition amount of conversion substrate being 0.10 to 1.00 g / L.

9. The method according to claim 4, characterized in that, The induction pre-culture conditions are as follows: inoculum size 2%–10% (v / v), culture temperature 30–37℃, and culture time 6–18 h.

10. The method according to claim 4, characterized in that, The fermentation temperature is 28–37°C, and the pH during the fermentation process is controlled between 4.0 and 6.0.

Images