A strain of poriazzns-y2 and application
The preparation of Crocusatin C and jasminodiol by fermentation with *Cyclocarya spp.* ZZNS-Y2 solves the problem of plant resource limitations in the preparation of compounds in existing technologies, and achieves efficient preparation of compounds and easy scale-up of production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QUJING NORMAL UNIV
- Filing Date
- 2025-06-10
- Publication Date
- 2026-07-03
AI Technical Summary
In existing technologies, the preparation of Crocusatin C and jasminodiol mainly relies on plant extraction, which is limited by plant resources and difficult to produce on a large scale.
Crocusatin C and jasminodiol were prepared by fermentation using a strain of *Caryopsis verticillata* ZZNS-Y2. The mixture was cultured in potato glucose seed medium and then fermented in solid fermentation media such as corn, potato, and rice. The extracts were separated and purified by silica gel column chromatography and high performance liquid chromatography.
It achieves efficient preparation of compounds, avoids plant resource limitations, is easy to scale up production, and the method is mild and pollution-free.
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Figure CN120843285B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a strain of *Carya spp.* ZZNS-Y2 and its applications, belonging to the field of microbial application technology. Background Technology
[0002] Crocusatin C is a compound mainly derived from saffron ( Crocus sativus ), dense flower ( Buddleja officinalis Gardenia ( Gardenia jasminoides Monoterpenoids from fruits, etc., possess biological activities such as anti-inflammatory and tyrosinase inhibitory activities. Jasminodiol, another monoterpenoid isolated from gardenia fruit, exhibits significant tyrosinase inhibitory and renal cell protective activities. Crocusatin C and jasminodiol both have potential applications in skin whitening and anti-aging. Currently, the preparation of Crocusatin C and jasminodiol is based on extraction and isolation from plants; no microbial fermentation preparation methods have been found, and the preparation of these compounds is limited by the availability of plant raw materials. Summary of the Invention
[0003] To address the problems existing in the preparation of Crocusatin C and jasminodiol in the prior art, one objective of this invention is to provide a strain of *Cyclocarya spp.* ZZNS-Y2, which was deposited at the China Center for Type Culture Collection (CCTCC) on July 15, 2024, with accession number CCTCC NO: M 20241561, and its taxonomic name is: Daldinia sp.; The address of the deposit is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
[0004] Another object of the present invention is to provide the application of *Crocusatin C* ZZNS-Y2 in the preparation of compounds Crocusatin C and jasminodiol.
[0005] The steps for preparing compounds Crocusatin C and jasminodiol using *Caryopsis rotundifolia* ZZNS-Y2 are as follows:
[0006] (1) The seed culture solution was obtained by inoculating the ZZNS-Y2 charcoal shell bacteria into potato glucose seed culture medium.
[0007] (2) The obtained seed culture solution is inoculated into the fermentation medium and fermented to obtain fermented microbial material.
[0008] (3) Soak, extract and filter the obtained fermentation bacteria in methanol solution to obtain filtrate, and evaporate the filtrate to obtain crude extract.
[0009] (4) A silica gel column was used to perform gradient elution separation of the crude extract using dichloromethane-methanol as the eluent. The volume ratio of dichloromethane to methanol was 30:1, 20:1, and 10:1. The eluents eluted at 20:1 were combined and concentrated to obtain concentrate a, and the eluents eluted at 10:1 were combined and concentrated to obtain concentrate b.
[0010] (5) The concentrate a was purified by gel column chromatography and concentrated to obtain crocusatin C.
[0011] (6) Jasminodiol was prepared by high performance liquid chromatography from concentrate b.
[0012] Preferably, the culture conditions in step (1) are 25℃~28℃ for 5~6 days.
[0013] Preferably, the fermentation medium in step (2) is one of corn solid fermentation medium, potato solid fermentation medium, corn grits solid fermentation medium or rice solid fermentation medium.
[0014] Preferably, the formula for corn solid fermentation medium is: 20±5g dried corn grits, 30±5mL water; the formula for potato solid fermentation medium is: 20±5g dry potato, 30±5mL water; the formula for corn grits solid fermentation medium is: 20±5g corn grits, 30±5mL water; and the formula for rice solid fermentation medium is: 20±5g rice, 30±5mL water.
[0015] Preferably, the fermentation culture conditions in step (2) are 28℃~37℃ for 30~40 days.
[0016] Preferably, the concentration method in step (4) is rotary evaporation.
[0017] Preferably, the eluent for gel column chromatography in step (5) is methanol, and the gel column used is Sephadex LH-20.
[0018] Preferably, in step (5), the high performance liquid chromatography uses a reversed-phase C18 column and acetonitrile as phase A and water as phase B as the mobile phase for gradient elution. The elution program is as follows: 0~5min: 5%A; 5~20min: 30%A; 20~35min: 70%A; 35~40min: 95%A; and the detection wavelength is 241nm.
[0019] The technical solution provided by this invention may include the following beneficial effects:
[0020] (1) This invention has discovered a new strain of crocusatin C and jasminodiol, ZZNS-Y2, which can simultaneously prepare compounds.
[0021] (2) The present invention utilizes microorganisms to prepare compounds crocusatin C and jasminodiol, which is not limited by plant resources and is easy to scale up production.
[0022] (3) The method described in this invention is efficient, pollution-free, and has mild reaction conditions. Attached Figure Description
[0023] Figure 1 For the fermentation product crocusatin C 13 C NMR and DEPT spectra.
[0024] Figure 2 For the fermentation product crocusatin C 1 H NMR spectrum.
[0025] Figure 3 For the fermentation product jasminodiol 13 C NMR and DEPT spectra.
[0026] Figure 4 For the fermentation product jasminodiol 1 H NMR spectrum.
[0027] Figure 5 The structural formulas for the fermentation products crocusatin C and jasminodiol are shown. Detailed Implementation
[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the content described.
[0029] The *Carya spp.* ZZNS-Y2 used in this example was isolated and screened from gardenia leaves. The ITS sequencing results of *Carya spp.* ZZNS-Y2 are shown in SEQ ID NO: 01.
[0030] Initially, *Carya chamaezia* ZZNS-Y2 appears as a white filamentous fungus on agar plates, gradually turning into gray filaments over time, while the reverse side of the plate turns grayish-black.
[0031] Preparation of different culture media:
[0032] (1) Corn solid fermentation medium: 20g dried corn grits, 30mL water.
[0033] (2) Potato solid fermentation culture medium: 20g dry weight potatoes, 30mL water.
[0034] (3) Rice solid fermentation culture medium: 20g rice, 30mL water.
[0035] (4) Corn grits solid fermentation culture medium: 20g corn grits, 30mL water.
[0036] Example 1
[0037] A method for preparing crocusatin C and jasminodiol by fermentation using the above-mentioned *Caryopsis buergerianum* ZZNS-Y2 includes the following steps:
[0038] (1) Inoculate ZZNS-Y2 of the genus ZZNS-Y2 into potato glucose seed culture medium at an inoculation rate of 5% and culture at 28°C for 5 days to obtain the seed culture solution.
[0039] (2) The obtained seed culture solution was inoculated into corn solid fermentation medium (20g dried corn grits and 30mL water) at an inoculation rate of 10%, and fermented at 28℃ for 30 days to obtain fermentation microbiota (including culture medium, mycelium and metabolites).
[0040] (3) The obtained fermentation bacteria were extracted by soaking in 50 mL of methanol solution and filtered to obtain filtrate. The filtrate was dried at 50 °C using a rotary evaporator to obtain crude extract.
[0041] (4) The crude extract was separated by gradient elution using a silica gel column with dichloromethane-methanol as the eluent. The volume ratio of dichloromethane to methanol was 30:1, 20:1, and 10:1. The eluents eluted at 20:1 were combined and evaporated at 50°C to obtain concentrate a. The eluents eluted at 10:1 were combined and evaporated at 50°C to obtain concentrate b. Concentrate a was purified by Sephadex LH-20 gel column chromatography (methanol as the eluent) and concentrated to obtain crocusatin. C; Jasminodiol was prepared by high performance liquid chromatography (HPLC) using a reversed-phase C18 column and a gradient elution of acetonitrile (phase A)-water (phase B). The elution program was as follows: 0-5 min: 5% A; 5-20 min: 30% A; 20-35 min: 70% A; 35-40 min: 95% A. The detection wavelength was 241 nm. The Rf values of the two compounds developed by dichloromethane-methanol (20:1) thin-layer chromatography were 0.55 and 0.40, respectively.
[0042] The calculated mass of crocusatin C was 38 mg, and the mass of jasminodiol was 60 mg. 13 C NMR (see) Figure 1 and 3 )and 1 H NMR (see) Figure 2 and 4The compounds were identified as crocusatin C and jasminodiol, respectively. Their chemical structures are shown in the appendix. Figure 5 .
[0043] Example 2
[0044] This invention provides a method for preparing crocusatin C and jasminodiol from the genus Daldinia sp. ZZNS-Y2 via fermentation, comprising the following steps:
[0045] (1) Daldinia sp. ZZNS-Y2 was inoculated into potato glucose seed culture medium at a rate of 5% and cultured at 28°C for 5 days to obtain the seed culture solution.
[0046] (2) The obtained seed culture solution was inoculated into potato solid fermentation medium (20g dry weight potato, 30mL water) at an inoculation rate of 10%, and fermented at 28℃ for 30 days to obtain fermentation microbiota (including culture medium, mycelium and metabolites).
[0047] (3) The obtained fermentation bacteria were soaked in 50 mL of methanol solution, extracted and filtered to obtain filtrate. The filtrate was dried at 50 °C using a rotary evaporator to obtain crude extract.
[0048] (4) The crude extract was separated by gradient elution using a silica gel column with dichloromethane-methanol as the eluent. The volume ratio of dichloromethane to methanol was 30:1, 20:1, and 10:1. The eluents eluted at 20:1 were combined and evaporated at 50°C to obtain concentrate a. The eluents eluted at 10:1 were combined and evaporated at 50°C to obtain concentrate b. Concentrate a was purified by Sephadex LH-20 gel column chromatography (methanol as the eluent) and concentrated to obtain crocusatin. C; Jasminodiol was prepared by high performance liquid chromatography (HPLC) using a reversed-phase C18 column and a gradient elution of acetonitrile (phase A)-water (phase B). The elution program was as follows: 0~5 min: 5% A; 5~20 min: 30% A; 20~35 min: 70% A; 35~40 min: 95% A; the detection wavelength was 241 nm.
[0049] The calculated mass of crocusatin C was 30 mg, and the mass of jasminodiol was 50 mg. Thin-layer chromatography (TLC) analysis was performed using dichloromethane-methanol (20:1) development and sulfuric acid-ethanol reagent for color development. The Rf values and spot colors were the same as those of the compounds obtained in Example 1, and they were identified as crocusatin C and jasminodiol, respectively. See the appendix for their chemical structures. Figure 5.
[0050] Example 3
[0051] This invention provides a method for preparing crocusatin C and jasminodiol from the genus Daldinia sp. ZZNS-Y2 via fermentation, comprising the following steps:
[0052] (1) Daldinia sp. ZZNS-Y2 was inoculated into potato glucose seed culture medium at a rate of 5% and cultured at 25°C for 6 days to obtain the seed culture solution.
[0053] (2) The obtained seed culture solution was inoculated into rice solid fermentation medium (20g rice, 30mL water) at an inoculation rate of 10%, and fermented at 28℃ for 30 days to obtain fermentation microbiota (including culture medium, mycelium and metabolites).
[0054] (3) The obtained fermentation bacteria were soaked in 50 mL of methanol solution, extracted and filtered to obtain filtrate. The filtrate was evaporated at 50 °C using a rotary evaporator to obtain crude extract.
[0055] (4) The crude extract was separated by gradient elution using a silica gel column with dichloromethane-methanol as the eluent. The volume ratio of dichloromethane to methanol was 30:1, 20:1, and 10:1. The eluents eluted at 20:1 were combined and evaporated at 50°C to obtain concentrate a. The eluents eluted at 10:1 were combined and evaporated at 50°C to obtain concentrate b. Concentrate a was purified by Sephadex LH-20 gel column chromatography (methanol as the eluent) and concentrated to obtain crocusatin. C; Jasminodiol was prepared by high performance liquid chromatography (HPLC) using a reversed-phase C18 column and a gradient elution of acetonitrile (phase A)-water (phase B). The elution program was as follows: 0~5 min: 5% A; 5~20 min: 30% A; 20~35 min: 70% A; 35~40 min: 95% A; the detection wavelength was 241 nm.
[0056] The calculated mass of crocusatin C was 24 mg, and the mass of jasminodiol was 45 mg. Thin-layer chromatography (TLC) analysis was performed using dichloromethane-methanol (20:1) development and sulfuric acid-ethanol reagent. The Rf values and spot colors were the same as those of the compounds obtained in Example 1, and they were identified as crocusatin C and jasminodiol, respectively. Their chemical structures are shown in the appendix. Figure 5 .
[0057] Example 4
[0058] This invention provides a method for preparing crocusatin C and jasminodiol from the genus Daldinia sp. ZZNS-Y2 via fermentation, comprising the following steps:
[0059] (1) The above-mentioned Daldinia sp. ZZNS-Y2 was inoculated into potato glucose seed culture medium at an inoculation rate of 5% and cultured at 28°C for 5 days to obtain the seed culture solution.
[0060] (2) The obtained seed culture solution was inoculated into corn grits solid fermentation medium (20g corn grits, 30mL water) at an inoculation rate of 10%, and fermented at 37℃ for 30 days to obtain fermentation microbiota (including culture medium, mycelium, and metabolites).
[0061] (3) The obtained fermentation bacteria were soaked in 50 mL of methanol solution, extracted and filtered to obtain filtrate. The filtrate was dried at 50 °C using a rotary evaporator to obtain crude extract.
[0062] (4) The crude extract was separated by gradient elution using a silica gel column with dichloromethane-methanol as the eluent. The volume ratio of dichloromethane to methanol was 30:1, 20:1, and 10:1. The eluents eluted at 20:1 were combined and evaporated at 50°C to obtain concentrate a. The eluents eluted at 10:1 were combined and evaporated at 50°C to obtain concentrate b. Concentrate a was purified by Sephadex LH-20 gel column chromatography (methanol as the eluent) and concentrated to obtain crocusatin. C; Jasminodiol was prepared by high performance liquid chromatography (HPLC) using a reversed-phase C18 column and a gradient elution of acetonitrile (phase A)-water (phase B). The elution program was as follows: 0~5 min: 5% A; 5~20 min: 30% A; 20~35 min: 70% A; 35~40 min: 95% A; the detection wavelength was 241 nm.
[0063] The calculated mass of crocusatin C was 35 mg, and the mass of jasminodiol was 63 mg. Thin-layer chromatography (TLC) analysis was performed using dichloromethane-methanol (20:1) development and sulfuric acid-ethanol reagent. The Rf values and spot colors were the same as those of the compounds obtained in Example 1, and they were identified as crocusatin C and jasminodiol, respectively. Their chemical structures are shown in the appendix. Figure 5 .
[0064] Example 5
[0065] This invention provides a method for preparing crocusatin C and jasminodiol from the genus Daldinia sp. ZZNS-Y2 via fermentation, comprising the following steps:
[0066] (1) Daldinia sp. ZZNS-Y2 was inoculated into potato glucose seed culture medium at a rate of 5% and cultured at 28°C for 5 days to obtain the seed culture solution.
[0067] (2) The obtained seed culture solution was inoculated into corn grits solid fermentation medium (20g corn grits, 30mL water) at an inoculation rate of 10%, and fermented at 28℃ for 40 days to obtain fermentation microbiota (including culture medium, mycelium, and metabolites).
[0068] (3) The obtained fermentation bacteria were soaked in 50 mL of methanol solution, extracted and filtered to obtain filtrate. The filtrate was dried at 50 °C using a rotary evaporator to obtain crude extract.
[0069] (4) The crude extract was separated by gradient elution using a silica gel column with dichloromethane-methanol as the eluent. The volume ratio of dichloromethane to methanol was 30:1, 20:1, and 10:1. The eluents eluted at 20:1 were combined and evaporated at 50°C to obtain concentrate a. The eluents eluted at 10:1 were combined and evaporated at 50°C to obtain concentrate b. Concentrate a was purified by Sephadex LH-20 gel column chromatography (methanol as the eluent) and concentrated to obtain crocusatin. C; Jasminodiol was prepared by high performance liquid chromatography (HPLC) using a reversed-phase C18 column and a gradient elution of acetonitrile (phase A)-water (phase B). The elution program was as follows: 0~5 min: 5% A; 5~20 min: 30% A; 20~35 min: 70% A; 35~40 min: 95% A; the detection wavelength was 241 nm.
[0070] The calculated mass of crocusatin C was 51 mg, and the mass of jasminodiol was 85 mg. Thin-layer chromatography (TLC) analysis was performed using dichloromethane-methanol (20:1) development and sulfuric acid-ethanol reagent for color development. The Rf values and spot colors were the same as those of the compounds obtained in Example 1, and they were identified as crocusatin C and jasminodiol, respectively. Their chemical structures are shown in the appendix. Figure 5 .
[0071] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.
Claims
1. A strain of *Carya argentea* ( Daldinia sp.) ZZNS-Y2, characterized in that: The accession number is CCTCC NO: M20241561.
2. The use of the *Caryota spp.* ZZNS-Y2 of claim 1 in the preparation of compounds crocusatin C and jasminodiol.
3. The application according to claim 2, characterized in that: The preparation steps are as follows: (1) The seed culture solution was obtained by inoculating the ZZNS-Y2 charcoal shell bacteria into potato glucose seed culture medium; (2) The obtained seed culture solution was inoculated into the fermentation medium and fermented to obtain fermented microbial material; (3) The obtained fermentation bacteria were soaked in methanol solution, extracted and filtered to obtain filtrate, and the filtrate was evaporated to obtain crude extract; (4) A silica gel column was used to perform gradient elution separation of the crude extract using dichloromethane-methanol as the eluent. The volume ratio of dichloromethane to methanol was 30:1, 20:1, and 10:
1. The eluents eluted at 20:1 were combined and concentrated to obtain concentrate a, and the eluents eluted at 10:1 were combined and concentrated to obtain concentrate b. (5) The concentrate a was purified by gel column chromatography and concentrated to obtain crocusatin C; (6) Jasminodiol was prepared by high performance liquid chromatography from concentrate b.
4. The application according to claim 3, characterized in that: The culture conditions in step (1) are 25℃~28℃ for 5~6 days.
5. The application according to claim 3, characterized in that: In step (2), the fermentation medium is one of corn solid fermentation medium, potato solid fermentation medium, corn grits solid fermentation medium or rice solid fermentation medium.
6. The application according to claim 5, characterized in that: The formula for corn solid-state fermentation medium is: 20±5 g dried corn grits, 30±5 mL water; the formula for potato solid-state fermentation medium is: 20±5 g dry potato, 30±5 mL water; the formula for corn grits solid-state fermentation medium is: 20±5 g corn grits, 30±5 mL water; the formula for rice solid-state fermentation medium is: 20±5 g rice, 30±5 mL water.
7. The application according to claim 3, characterized in that: The fermentation conditions in step (2) are 28℃~37℃ for 30~40 days.
8. The application according to claim 3, characterized in that: The concentration method in step (4) is rotary evaporation.
9. The application according to claim 3, characterized in that: In step (5), the eluent for gel column chromatography is methanol, and the gel column used is Sephadex LH-20.
10. The application according to claim 3, characterized in that: In step (6), high performance liquid chromatography uses a reversed-phase C18 column and gradient elution with acetonitrile as phase A and water as phase B. The elution program is as follows: 0~5 min: 5% A; 5~20 min: 30% A; 20~35 min: 70% A; 35~40 min: 95% A; detection wavelength is 241 nm.