A method of preparing equol for delaying ovarian aging

Abstract

The present application relates to the field of biosynthesis, and specifically discloses a preparation method of equol for delaying ovarian aging, which realizes co-fermentation through fat bean powder and a composite starting substrate; a binary fermentation system constructed by matching auxiliary strains and a specially prepared fermentation medium, so that integrated optimization of the extraction process is realized.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis, specifically relating to a method for preparing equadol for delaying ovarian aging. Background Technology

[0002] Estrol belongs to the isoflavone class of compounds. Its molecular core consists of two benzene rings connected by an oxygen-containing heterocycle (chromium ring). It exists in two enantiomers, S-type and R-type. It is not a direct dietary component, but a key metabolic end product of soy isoflavones under the action of intestinal microorganisms. It has excellent estrogen receptor affinity and antioxidant capacity.

[0003] Early synthetic routes for equol typically started with specific flavonoids, proceeding through a series of chemical reactions to obtain mixed equol (i.e., a mixture of R- and S-forms), and finally, through an additional chiral resolution step, yielded a single enantiomer with high physiological activity. However, due to policy and regulatory pursuits for efficient, economical, and green industrial production, the application of chemical synthesis methods has been greatly limited.

[0004] Biosynthesis is a production method that utilizes microorganisms or their produced enzymes to convert substrates into the target product, equol. Currently, researchers have screened various strains from the environment capable of efficiently converting soybean isoflavones into equol. For example, Acinetobacter AUH-JLM455, discovered in patent CN101338294A, can convert daidzein or daidzein into S-equol under anaerobic conditions, and its inhibitory effect on liver cancer cells has been confirmed. The study "Microbial Degradation of Isoflavones in Soybean Meal and Extraction of Degradation Products" indicates that strain ZX-7, after fermenting soybean meal under optimized conditions for 72 hours, can accumulate 3.39 mg of equol per 100 g of soybean meal. Although microbial fermentation is a mild method, traditional intestinal bacteria are typically strict anaerobes, requiring demanding cultivation equipment and environmental conditions, resulting in low production efficiency and difficulty in meeting the needs of large-scale production.

[0005] In view of the problem of low production efficiency of existing microbial fermentation methods, the present invention aims to provide a method for preparing equol with high production efficiency and easy separation. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a highly efficient biosynthetic preparation method for equol based on natural microbial strains. This method utilizes defatted soybean flour and a composite starter substrate for co-fermentation. By combining a binary fermentation system constructed with auxiliary strains and a specially formulated fermentation medium, the extraction process is integrated and optimized.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing equadol for delaying ovarian aging, specifically comprising the following steps: S1. The freeze-dried main and secondary bacterial cultures were inoculated into BHI solid medium and cultured under anaerobic conditions at 30-40℃ for 24-48h. Then, single colonies were picked and inoculated into BHI liquid medium and cultured statically under anaerobic conditions at 30-40℃ for 12-36h to obtain the main seed culture and secondary seed culture, respectively. S2. Inoculate the main seed culture at an inoculation rate of 8-12 wt% into the fermentation medium and culture it under anaerobic conditions at 30-40℃ for 1-24 h. Then add defatted soybean flour and macroporous resin, and culture for 24-36 h. Inoculate the secondary seed culture at an inoculation rate of 4-6 wt% into the fermentation medium and continue to culture for 24-36 h. Take samples every 4-8 h to monitor the equadol content, and filter to obtain the bacterial mixture and resin. S3. Soak the resin in ethanol, shake and elute for 2-4 hours, collect the ethanol eluent, extract the eluent with an equal volume of solvent, combine the organic phases, and distill under reduced pressure to obtain crude equadol; purify the crude product using preparative liquid chromatography to obtain equadol.

[0008] In some embodiments, in step S1, the main bacterial species is Acinetobacter AUH-JLM455 and the secondary bacterial species is Clostridium dihydrogenus ZX-7-9.

[0009] In some embodiments, in step S2, the ratio of defatted soybean flour to fermentation culture medium is 40g-70g:1L.

[0010] The first aspect of this application is to improve substrate utilization by supplementing defatted soybean flour rich in daidzein during the middle stage of fermentation and utilizing the β-glucosidase activity secreted by the strain itself to achieve a cascade reaction of simultaneous hydrolysis and conversion. Compared with the traditional method that uses high-cost pure daidzein as substrate, this reduces cost input.

[0011] The second aspect uses Acinetobacter AUH-JLM455 (CGMCC No. 2333) as the main strain to directionally convert the substrate into pure S-estrol. It is combined with the introduction of the auxiliary strain Clostridium dihydrogenase ZX-7-9 (CGMCC NO. 1995) in the later stage of fermentation to construct a binary fermentation system. By utilizing the metabolic preferences of different strains for intermediate products, the product feedback inhibition can be relieved.

[0012] Thirdly, by adding macroporous adsorption resin to the fermenter, the generated equol is quickly adsorbed by the resin, avoiding the inhibitory effect of the product on the metabolism of the strain, thereby improving the overall conversion rate.

[0013] In some embodiments, in step S2, the fermentation medium is a mixture of a basic carbon source, a complex nitrogen source, a buffer salt, a starter substrate, and purified water.

[0014] In some embodiments, the concentrations of the basic carbon source, the complex nitrogen source, the buffer salt, and the initiator substrate in the fermentation medium are 3-7 g / L, 12-18 g / L, 4-8 g / L, and 180-240 mg / L, respectively.

[0015] In some embodiments, the base carbon source comprises glucose and cellobiose and / or soybean oligosaccharides.

[0016] Preferably, the basic carbon source comprises glucose, cellobiose, and / or soybean oligosaccharides in a mass ratio of (1-3):(1-3):(0.1-0.9).

[0017] During the 0-24h cell growth phase, glucose provides rapid initial energy, and the strain utilizes glucose for rapid proliferation. This invention avoids metabolic inhibition (Crabtree effect) that may be caused by high glucose concentrations through glucose concentration control. Cellobiose, as a slowly utilized carbon source, can prolong the logarithmic growth phase of the strain and can also act as an inducer of cellulase, providing pre-adaptation and enzyme-inducing signals for the strain to synergistically utilize the carbon source in the subsequently added cellulose-rich defatted soybean flour. Soybean oligosaccharides directly mimic natural substrates in the intestinal environment and can activate dormant genes in the strain related to soybean aglycone transport or metabolism, achieving substrate induction.

[0018] In some embodiments, the buffer salt comprises KHCO3 and MgSO4·7H2O.

[0019] Traditional Na + and Cl - Basic salts can cause osmotic stress and ion toxicity. The KHCO3 used in this invention provides K... + At the same time, HCO3 - It can also act as a natural pH buffer to improve the simulation of the intestinal physiological environment; compared to blindly adding complex salts, the Mg in specifically selected MgSO4·7H2O 2+ As a cofactor for ATPase and partial reductase, it can provide the necessary trace elements for the enzymatic reaction of estrol production, and is more suitable for this invention.

[0020] In some embodiments, the initiating substrate comprises daidzein and soybean lecithin.

[0021] In some embodiments, the soybean lecithin contains ≥70% phosphatidylcholine.

[0022] Soybean lecithin can form inclusion complexes or mixed micelles with hydrophobic daidzein, improving the solubility and dispersibility of daidzein in aqueous culture media, making it easier for bacterial cells to take up. The choline and phosphatidyl groups provided by lecithin can serve as direct precursors for bacterial cell membrane synthesis, reducing the energy consumption of cell membrane synthesis under stress and repairing damaged membrane structures, resulting in stronger cell membranes. Under the osmotic pressure and toxicity stresses of late-stage anaerobic fermentation and product accumulation, it can maintain a more stable cell membrane structure and function, thus preserving higher activity and conversion efficiency.

[0023] In some embodiments, the composite nitrogen source comprises tryptone and yeast extract.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention improves substrate utilization by supplementing defatted soybean flour rich in daidzein during the fermentation process, utilizing the β-glucosidase activity secreted by the bacterial strain to achieve a cascade reaction of simultaneous hydrolysis and conversion. Simultaneously, the use of daidzein and soybean lecithin as starter substrates enhances the solubility and dispersibility of daidzein in the aqueous culture medium, making it easier for bacterial cells to take up. Furthermore, soybean lecithin helps maintain a more stable cell membrane structure and function, thus preserving higher activity and conversion efficiency. Compared to traditional methods that use expensive pure daidzein as a substrate, this invention reduces cost input.

[0025] 2. This invention uses Acinetobacter AUH-JLM455 (CGMCC No. 2333) as the main strain, and introduces the auxiliary strain Clostridium dihydrogenase ZX-7-9 (CGMCC NO. 1995) in the later stage of fermentation to construct a binary fermentation system. By utilizing the metabolic preferences of different strains for intermediate products, the product feedback inhibition is relieved. At the same time, by adding macroporous adsorption resin to the fermenter, the generated equol is quickly adsorbed by the resin, avoiding the inhibitory effect of the product on the metabolism of the strain and improving the overall conversion rate.

[0026] 3. In the preparation of fermentation medium, this invention introduces a functional carbon source to achieve stress resistance and induction effect; at the same time, it abandons the practice of blindly adding or avoiding inorganic salts, and achieves precise supply of trace elements through ion regulation; and upgrades the simple chemical reagent substrate daidzein to a complex substrate system with synergistic effects to improve the overall conversion efficiency. Detailed Implementation

[0027] The present invention will be described below with reference to specific implementation schemes. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. It is worth noting that, unless otherwise specified, the raw materials used in the following preparation examples and embodiments can be from any commercially available manufacturer.

[0028] Preparation Example 1 The preparation steps for fermentation medium A are as follows: Fermentation medium A is obtained by mixing the basic carbon source (3g glucose, 2g cellobiose, 0.5g soybean oligosaccharide), the complex nitrogen source (10g tryptone, 5g yeast extract), the buffer salt (2.5g KHCO3 and 0.1g MgSO4·7H2O), the starting substrate (10mg daidzein, 200mg soybean lecithin with 80% phosphatidylcholine content) and 1L purified water.

[0029] Preparation Example 2 The preparation steps for fermentation medium B are as follows: Fermentation medium B is obtained by mixing the basic carbon source (5g glucose), the complex nitrogen source (10g tryptone, 5g yeast extract), the buffer salt (2.5g KHCO3 and 0.1g MgSO4·7H2O), the starting substrate (10mg daidzein, 200mg soybean lecithin with 80% phosphatidylcholine content) and 1L purified water.

[0030] Preparation Example 3 The preparation steps for fermentation medium C are as follows: Fermentation medium C is obtained by mixing the basic carbon source (3g glucose, 2g cellobiose, 0.5g soybean oligosaccharide), the complex nitrogen source (10g tryptone, 5g yeast extract), the buffer salt (2.5g KHCO3 and 0.1g MgSO4·7H2O), the starting substrate (50mg daidzein), and 1L purified water.

[0031] Example 1 A method for preparing equadol for delaying ovarian aging specifically includes the following steps: S1. The freeze-dried Acinetobacter AUH-JLM455 and Clostridium dihydrogen ether ZX-7-9 were inoculated into BHI solid medium and cultured under anaerobic conditions at 37℃ for 36h. Then, single colonies were picked and inoculated into BHI liquid medium and cultured under anaerobic conditions at 37℃ for 24h to obtain the main seed culture and the secondary seed culture. S2. Inoculate the main seed culture into fermentation medium A at an inoculation rate of 10 wt% and culture under anaerobic conditions at 37℃ for 12 h. Then add defatted soybean flour (60 g / L fermentation medium) and XAD-7 macroporous resin (50 g / L fermentation medium) and culture for 36 h. Inoculate the secondary seed culture into fermentation medium at an inoculation rate of 5 wt% and continue to culture for 24 h. Take samples every 6 h to monitor the equadol content and filter to obtain the bacterial mixture and resin. S3. Soak the resin with an equal weight of 95% ethanol, shake and elute for 2 hours, collect the ethanol eluent, extract the eluent three times with an equal volume of ethyl acetate, combine the organic phases, and distill under reduced pressure at 40℃ to obtain crude equadol. Purify the crude product using preparative liquid chromatography with a methanol-water system as the mobile phase to obtain equadol.

[0032] Example 2 This embodiment provides a method for preparing estrol for delaying ovarian aging. The specific implementation method is the same as in Embodiment 1, except that fermentation medium A is replaced by an equal amount of fermentation medium B.

[0033] Example 3 This embodiment provides a method for preparing estrol for delaying ovarian aging. The specific implementation method is the same as in Embodiment 1, except that fermentation medium A is replaced by an equal amount of fermentation medium C.

[0034] Comparative Example 1 A method for preparing equadol for delaying ovarian aging specifically includes the following steps: S1. The freeze-dried Acinetobacter AUH-JLM455 strain was inoculated into BHI solid medium and cultured under anaerobic conditions at 37℃ for 36h; then a single colony was picked and inoculated into BHI liquid medium and cultured statically under anaerobic conditions at 37℃ for 24h to obtain the seed culture. S2. Inoculate the seed culture into fermentation medium A at an inoculation rate of 10 wt% and culture it under anaerobic conditions at 37°C for 12 h. Then add defatted soybean flour (60 g / L fermentation medium) and XAD-7 macroporous resin (50 g / L fermentation medium) and culture for 60 h. Take samples every 6 h to monitor the estrol content and filter to obtain the bacterial culture mixture and resin. S3. Soak the resin in 95% ethanol, shake and elute for 2 hours, collect the ethanol eluent, extract the eluent three times with an equal volume of ethyl acetate, combine the organic phases, and distill under reduced pressure at 40℃ to obtain crude equadol. Purify the crude product using preparative liquid chromatography with a methanol-water system as the mobile phase to obtain equadol.

[0035] Performance testing Each test group had 3 parallel samples. The specific results are shown in Table 1.

[0036] 1. Cell membrane integrity test: 24 hours after adding defatted soybean flour, take 50 mL of fermentation broth, centrifuge at 4℃ (10000 rpm, 10 min), collect the supernatant, and use the LDH cytotoxicity assay kit to measure the absorbance at 492 nm. LDH is an intracellular enzyme that leaks into the culture medium when the cell membrane is damaged. The higher the LDH activity in the supernatant, the more severe the membrane damage.

[0037] 2. Enzyme activity test: Before adding soybean flour, take 50 mL of fermentation broth, centrifuge at 4℃ to collect cells (10000 rpm, 10 min), wash twice with 0.1 M phosphate buffer (pH 7.0), and resuspend in 5 mL buffer; sonicate to disrupt cells (ice bath, 200 W, 3 s working / 5 s rest, 10 min total); centrifuge at 4℃ (12000 rpm, 20 min), the supernatant is the crude enzyme solution, and the protein concentration in the crude enzyme solution is determined by the Bradford method; using p-nitrophenyl-β-D-glucoside (pNPG) as substrate, measure the rate of increase in absorbance at 405 nm, and calculate the specific activity of β-glucosidase (U / mg protein); one enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute.

[0038] 3. Estrol yield test: Take 5 mL of the ethanol eluent from step S3 and detect the concentration by HPLC. The detection wavelength is 280 nm, and the mobile phase is methanol:water = 55:45 (V / V).

[0039] Table 1 As shown in Table 1, the final equol concentration in Example 1 reached over 550 mg / L, which may be attributed to the membrane protective effect of lecithin and the elimination of reaction inhibition. In the later stages of fermentation, the LDH release in Example 3 was significantly higher than that in Example 1, indicating a decrease in the proportion of viable bacteria. This suggests that the addition of soybean lecithin to the initiating substrate enhanced cell membrane integrity. Combining Examples 2 and 1, it can be seen that the induction of cellobiose may promote an increase in the specific activity of β-glucosidase, demonstrating the inducing effect of cellobiose and soybean oligosaccharides in the complex carbon source on enzymes related to daidzein metabolism. Compared to Example 1, the equol yield in Comparative Example 1 decreased, possibly due to incomplete daidzein conversion caused by the lack of synergistic effect of a single strain.

[0040] The embodiments described above do not limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations 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 scope of the present invention.

Claims

1. A method for preparing equadol for delaying ovarian aging, characterized in that, Specifically, it includes the following steps: S1. The freeze-dried main and secondary bacterial cultures were inoculated into BHI solid medium and cultured under anaerobic conditions at 30-40℃ for 24-48h. Then, single colonies were picked and inoculated into BHI liquid medium and cultured statically under anaerobic conditions at 30-40℃ for 12-36h to obtain the main seed culture and secondary seed culture, respectively. S2. Inoculate the main seed culture at an inoculation rate of 8-12 wt% into the fermentation medium and culture it under anaerobic conditions at 30-40℃ for 1-24 h. Then add defatted soybean flour and macroporous resin, and culture for 24-36 h. Inoculate the secondary seed culture at an inoculation rate of 4-6 wt% into the fermentation medium and continue to culture for 24-36 h. Take samples every 4-8 h to monitor the equadol content, and filter to obtain the bacterial mixture and resin. S3. Soak the resin in ethanol, shake and elute for 2-4 hours, collect the ethanol eluent, extract the eluent with an equal volume of solvent, combine the organic phases, and distill under reduced pressure to obtain crude equadol; purify the crude product using preparative liquid chromatography to obtain equadol.

2. The method for preparing equadol according to claim 1, characterized in that, In step S1, the main bacterial species is Acinetobacter AUH-JLM455 and the secondary bacterial species is Clostridium dihydrogenus ZX-7-9.

3. The method for preparing equadol according to claim 1, characterized in that, In step S1, the ratio of defatted soybean flour to fermentation culture medium is 40g-70g:1L.

4. The method for preparing equadol according to claim 1, characterized in that, In step S2, the fermentation medium is composed of a basic carbon source, a complex nitrogen source, a buffer salt, a starter substrate, and purified water.

5. The method for preparing equadol according to claim 4, characterized in that, The concentrations of the basic carbon source, complex nitrogen source, buffer salt, and initiator substrate in the fermentation medium are 3-7 g / L, 12-18 g / L, 4-8 g / L, and 180-240 mg / L, respectively.

6. The method for preparing equadol according to claim 4, characterized in that, The basic carbon source comprises glucose and cellobiose and / or soybean oligosaccharides.

7. The method for preparing equadol according to claim 4, characterized in that, The buffer salt contains KHCO3 and MgSO4·7H2O.

8. The method for preparing equadol according to claim 4, characterized in that, The initiating substrates include daidzein and soybean lecithin.

9. The method for preparing equadol according to claim 8, characterized in that, The soybean lecithin contains ≥70% phosphatidylcholine.

10. The method for preparing equadol according to claim 4, characterized in that, The composite nitrogen source comprises tryptone and yeast extract.

Images