Fusarium oxysporum and application thereof in preparation of yuccone

Abstract

The invention belongs to the technical field of microorganisms, and relates to fusarium oxysporum and application thereof in preparation of yuccone. The fusarium oxysporum disclosed by the invention has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.40905. Yellow ginger can be converted into yuccone by utilizing the fusarium oxysporum under proper fermentation conditions. The invention develops a biotransformation production process for industrially preparing the yuccone, and the production cost of the yuccone is effectively reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Fusarium oxysporum and application thereof in preparing syringone. Background Art

[0002] Smilagenone belongs to the class of sapogenin derivative compounds, and its English name is Smilagenone (Chakravarti, Chakravarti et al. 1960). At present, many scholars have found that this compound and its derivatives have good efficacy in treating Alzheimer's disease (AD). Although significant progress has been made in the study of AD in recent years, its pathological mechanism has not yet been fully elucidated. A large number of studies have shown that oxidative stress (OS) plays a key role in the development of AD. In the study of AD, natural products have received widespread attention due to their potential neuroprotective activity. Specifically, these compounds can protect neurons from oxidative stress by inhibiting the generation of ROS / RNS and reducing intracellular oxidative damage (Zhang, Wu et al. 2019, Pereira Moreno, Mantovanelli et al. 2020). These findings provide valuable clues for the development of new neuroprotective agents, especially in the treatment of Alzheimer's disease. Further in-depth research on the relationship between the structure and activity of these derivatives may lead to more effective drugs for the treatment of AD. In addition, some scholars have studied the relationship between syringone and human cancer cells. These studies used syringone to experiment on cancer cells in the human body, and ultimately concluded that syringone can induce apoptosis of cancer cells (Elekofehinti, Iwaloye et al. 2021). In addition, syringone has a positive effect on immune regulation, cardiovascular function, blood lipid regulation, virus resistance, etc., and is widely used in related drug development. The preparation method of syringone currently relies on isolating it from the rhizome of Dioscorea zingiberensis through low-melting point diosgenin, and lacks a large-scale preparation route. Summary of the invention

[0003] The purpose of the present invention is to provide a Fusarium oxysporum and its application in the preparation of yucanone. The Fusarium oxysporum of the present invention can convert turmeric into yucanone, which can effectively reduce the production cost of yucanone and provide a biological basis for developing a bioconversion production process for industrial production of yucanone.

[0004] The Fusarium oxysporum of the present invention is isolated from the rhizome of Dioscorea zingiberensis, and the preservation information is as follows.

[0005] Strain name: SY fxl 23.3;

[0006] Classification name: Fusarium oxysporum;

[0007] Date of receipt by the depository: October 27, 2023;

[0008] Time of issuance of preservation certificate: December 27, 2023;

[0009] Depository: China General Microbiology Center (CGMCC);

[0010] Deposit number: CGMCC No.40905.

[0011] The Fusarium oxysporum of the present invention produces syranone by fermentation under the culture condition containing turmeric.

[0012] The present invention also claims to protect the use of the Fusarium oxysporum in preparing yucanone.

[0013] As a preferred application of the present invention, the Fusarium oxysporum converts turmeric into yuccaone.

[0014] As a preferred application of the present invention, the Fusarium oxysporum produces syringone through solid-state fermentation.

[0015] As a preferred application of the present invention, the solid-state fermentation culture medium contains 70wt% turmeric and 4wt% yeast extract powder.

[0016] As a preferred application of the present invention, the conditions of the solid-state fermentation are: fermentation time 9 days, fermentation temperature 32°C.

[0017] As a preferred application of the present invention, the solid-state fermentation process is stirred and water is replenished; in terms of mass percentage, the water replenishment amount is 60% of the solid-state fermentation material.

[0018] As a preferred application of the present invention, the solid-state fermentation product of Fusarium oxysporum is extracted with petroleum ether as solvent, eluted with a silica gel column and purified by thin layer chromatography to obtain syringone.

[0019] As a preferred application of the present invention, the solid-state fermentation drying product of Fusarium oxysporum is extracted by mixing 1 kg of dry substrate with 5 L of petroleum ether with ultrasound assistance;

[0020] The extraction was assisted by ultrasound, and the ultrasonic parameter conditions were: ultrasonic frequency 99 Hz, and ultrasonic time 30 min.

[0021] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages.

[0022] An endophyte from Dioscorea zingiberensis was isolated and identified as F.oxysporum strain SY fxl 23.3. The Fusarium oxysporum can produce the compound syringone through solid-state fermentation. The compound has multiple pharmaceutical activities and is used in cancer treatment and Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solution of the present invention, the drawings involved in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the description are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is the colony morphology of Fusarium oxysporum strain after being cultured on PDA medium for 2 days.

[0025] Figure 2 This is the bacterial morphology of the Fusarium oxysporum strain after 5 days of fermentation.

[0026] Figure 3 This is a 100-fold magnification of the mycelium morphology of the Fusarium oxysporum strain after staining with lactic acid carbolic acid cotton blue.

[0027] Figure 4 This is the morphology of hyphae and spores of Fusarium oxysporum strain under a scanning electron microscope.

[0028] Figure 5 This is a solid-state fermentation reaction diagram.

[0029] Figure 6 This is a crystal diagram of syringone.

[0030] Figure 7 Silcanone 1 H hydrogen spectrum.

[0031] Figure 8 Silcanone 13 C carbon spectrum.

[0032] Fig. 9 This is the LC-MS / MS image of syringone.

[0033] Fig.10 This is the DEPT spectrum of syringone.

[0034] Fig.11 This is the HSQC spectrum of syringone.

[0035] Fig.12 It is the molecular formula of syringone.

[0036] Fig.13 This is the absolute configuration stereogram of syringone.

[0037] Fig.14 This is the absolute configuration plan of syringone.

[0038] Fig.15 This is the unit cell structure diagram of syringone. DETAILED DESCRIPTION

[0039] The technical scheme of the present invention is described below in conjunction with the examples, but the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified. The "%" indicates mass percentage unless otherwise specified.

[0040] Example 1

[0041] This example provides a strain of Fusarium oxysporum (F.oxysporum) SY fxl 23.3 Collection and separation.

[0042] Rinse the rhizomes of Dioscorea with water, place them on a clean bench, and cut them into 5 cm × 5 cm small pieces for later use; first soak the rhizomes of Dioscorea in 75% ethanol for 30 seconds, take them out and rinse them with sterile water for 4 times, then rinse them with 0.01% HgCl 2 Soak for 3 minutes, take out and rinse with sterile water for 4 times; after the above sterilization operation is completed, cut the rhizome of Dioscorea zingiberensis into a length of about 0.5 cm, inoculate it into a culture dish, inoculate 2 pieces in each culture dish, make 3 replicates, and place it in a 28°C incubator for 5 days; after colonies grow around the tissue block, pick the bacteria with an inoculation loop, streak on the PDA culture medium, seal and mark it, and then culture it in a 28°C constant temperature incubator; after colonies grow, repeat this step, streak repeatedly, and after obtaining a single colony, use PDA slant culture base to store at 4°C for standby use, and isolate the Fusarium oxysporum (F.oxysporum) strain SY fxl 23.3.

[0043] The present invention classifies and identifies the Fusarium oxysporum strain by the 16S rDNA sequence method. A person skilled in the art can easily extract the DNA of the Fusarium oxysporum and amplify the sequence of the 16S rDNA by referring to the existing technology. The gene sequence of the Fusarium oxysporum strain has been submitted to the GenBank database of NCBI, and the accession number of the Fusarium oxysporum strain on GenBank is: OR707928. The amplified product is subjected to Sanger bidirectional sequencing, and the sequencing results are compared and analyzed on NCBI, and the species with a homology of more than 97% and the highest homology is judged as the species of the strain. The sequencing results show that the similarity between the 16S rDNA gene sequence of the Fusarium oxysporum strain and the nearest effective strain is 99%.

[0044] In this example, the Fusarium oxysporum strain was classified and identified through morphological characteristics. Figure 1 This is a colony morphology diagram of the Fusarium oxysporum strain after being cultured on PDA medium for 2 days. Figure 2 This is a bacterial morphology diagram of the Fusarium oxysporum strain after 5 days of fermentation culture.

[0045] Figure 3 The mycelium morphology of the Fusarium oxysporum strain is magnified 100 times after being stained with lactic acid carbolic acid cotton blue. Figure 4 The morphological diagram of hyphae and spores of Fusarium oxysporum under a scanning electron microscope. The Fusarium oxysporum strain was inoculated on a PDA culture medium, and the morphological characteristics presented were: the strain grew rapidly, the colony diameter reached 1.5 cm, the texture was dense, the colony was flat, the initial white short fuzzy hyphae grew evenly around, the colony edge was fuzzy, and the color of the center of the colony gradually deepened to light purple in the later stage, and a large number of light purple spores grew; the aerial hyphae of the strain had septa. Combined with the strain morphological observation, culture characteristics and 16SrDNA identification results, the Fusarium oxysporum described in the present invention belongs to the genus Fusarium.

[0046] Example 2

[0047] This example provides a strain of Fusarium oxysporum (F.oxysporum) SY fxl 23.3 Activation and preparation process of syringone.

[0048] Fusarium oxysporum was inoculated on PDA medium for activation. 6.4 g sucrose, 2.2 g beef extract, 8 g yam rhizome powder, 0.5 g K 2 HPO 4 Dissolve 0.25g KCl in 1L of water, sterilize, and cool to room temperature to obtain a culture medium. Inoculate the activated strains on the above culture medium, and shake flask ferment for 36 hours (fermentation speed 150rmp, fermentation temperature 32°C) to obtain a first-level seed solution. Then inoculate the obtained first-level seed solution into the culture medium at an inoculum of 5% (the culture medium used is the same as the culture medium for preparing the first-level seed solution), and shake flask ferment for 24 hours (fermentation speed 150rmp, fermentation temperature 32°C) to obtain a second-level seed solution.

[0049] Take an appropriate amount of turmeric, crush it and pass it through a 100-mesh sieve to obtain a sieved powder. The sieved powder is inoculated in SY fxl 23.3 secondary seed liquid, inoculated at 70%, added 4% sucrose and beef extract (biological reagent, Beijing Aoboxing Biotechnology Co., Ltd.) and stirred evenly to obtain a culture medium. The culture medium was subjected to solid-state fermentation (fermentation temperature 32°C) for 9 days, and was fully stirred and 50% water was supplied during the fermentation process. The fermentation process was as follows Figure 5 shown.

[0050] After the fermentation is completed, the solid fermentation product is placed in an oven at 55°C for drying and then crushed. Then 4g of the crushed powder is sampled by a uniform method, and 20mL of petroleum ether is added and mixed, and then placed in a shaker with a rotation speed of 150rpm. After 30min and 99Hz ultrasonic extraction, the extract is collected in a plate. Repeat the above sampling and mixing petroleum ether extraction steps three times, collect the obtained extract in the same plate, and obtain the crude product of syringone after the collected liquid evaporates.

[0051] To the crude product of Dioscorea syringone, chloroform was added to dissolve it again, and the volume was fixed to 10 mL in a volumetric flask. After the liquid component in the obtained solution was completely volatilized, the crystals of syringone were obtained. The crystals of purified syringone were as follows: Figure 6 shown.

[0052] 9 kg of the product obtained by solid-state fermentation was extracted 5 times with 3 volumes of petroleum ether, and the petroleum ether was recovered by concentration under reduced pressure to finally obtain 55.38 g of petroleum ether extract.

[0053] Use a forward silica gel column to divide the section, petroleum ether and ethyl acetate as eluents, elution in a ratio of 15:1, and collect the eluent in a 250mL conical flask, 200mL per bottle. While separating, use TLC to detect whether syringone is eluted. When it is completely eluted, use 5L of petroleum ether and ethyl acetate (15:1) eluent in total, and collect 25 bottles of eluent. Use a rotary evaporator to spin dry the sample, weigh it, and obtain a sample containing syringone of 20.21g. Label, seal, and store at low temperature for later use.

[0054] The 20.21 g sample separated in 3.2 was mixed with C18 silica gel and a medium-pressure pump was used with a solvent of petroleum ether and ethyl acetate (10:1) as the eluent. The collected samples were detected by TCL, and the samples containing the target product were combined, spin-dried, and weighed to obtain 8.04 g of sample.

[0055] The sample was further purified by separation and purification using preparative thin layer chromatography to obtain 120 mg of pure product.

[0056] The crystals are white and blocky. The obtained compound was tested by mass spectrometry, hydrogen spectrum and carbon spectrum. Through comprehensive analysis of all spectral data, the structural formula of serpentine was obtained as follows: Fig.12 .

[0057] The molecular formula of this compound is C 27 H 42 O 3 ,exist 1In the H NMR spectrum, there are 4 methyl signals in the high field region δH 0.79 (3H, s), 1.03 (3H, s), 0.97 (3H, d, J = 6.9 Hz), 0.79 (3H, d, J = 6.3 Hz), 3 hydrogen signals connected to oxygen 4.41 (dd, 14.3, 7.9), 3.48 (ddd, 10.9, 4.5, 2.1), 3.37 (t, 10.9), 4 hydrogen signals connected to carbonyl 2.17 (m, 1H), 2.02 (m, 1H), 2.68 (dd, 15.1, 13.3 Hz, 1H, H-4a); 2.02 (m, 1H, H-4b). In the 1H NMR spectrum, the hydrogen signals at δH1-2 ppm overlap seriously. From this feature, it can be preliminarily inferred that the compound is an alicyclic compound, which may be a steroid. 13 The CNMR and DEPT135° spectra contain a total of 27 carbons, including 4 quaternary carbons (δC 213.4, 109.4, 40.8, 35.1), 8 methines (δC 80.9, 62.3, 56.4, 44.3, 41.7, 40.9, 35.3, 30.4), 11 methylenes (δC 67.0, 42.5, 40.2, 37.3, 37.1, 31.9, 31.5, 28.9, 26.6, 26.2, 21.1), and 4 methyls (δC 22.8, 17.3, 16.6, 14.6). By comparing the characteristic signals of this type of compound, the chemical shift of C-1 is δC 213.4ppm, C-22 is 109.4ppm, C-26 is assigned to 67.0ppm, and C-16 is assigned to 80.9ppm. In summary, the structure of this compound is determined to be Dioscorea serrone. Figure 7 , Figure 8 It is the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum. Fig. 9 This is the high-resolution mass spectrum of syringone. Fig.10 is the DEPT spectrum of syringone, Fig.11 This is the HSQC spectrum of syringone.

[0058] Example 3

[0059] This example provides a strain of Fusarium oxysporum (F.oxysporum) SY fxl 23.3 Single crystal diffraction analysis results of syringone were obtained.

[0060] The diffractometer was BRUKER D8 VENTURE equipped with a cryogenic device and a Cu target was used. The diffractometer was used to measure the crystal data at a test temperature of 100K. Data were collected in scanning mode. The structure was solved by direct method using SHELXT program, all data were integrated with SAINT, and multiple scan absorption correction was performed using SADABS. The structure was solved by SHELXT and refined by full matrix least squares method using Olex2 using XL for F2. Full matrix least squares method anisotropic refinement was performed on the coordinates and thermal vibration parameters of all non-hydrogen atoms. All hydrogen atoms were positioned by riding bridge mode and refined by isotropic method.

[0061] Unit cell structure: There are 4 H1 molecules in one unit cell, and the characteristic elements in the crystal include three 21-helical axes. Fig.15 .

[0062] Absolute configuration represents the true spatial arrangement relationship of each group in a chiral molecule, that is, the absolute spatial configuration arrangement relationship. Fig.13 and Fig.14 The absolute configuration of the unit cell molecular structure presented is that the molecule contains 11 chiral carbon atoms, and the absolute configurations of the chiral carbons are C5(R), C8(R), C9(S), C10(S), C13(S), C14(S), C16(S), C17(R), C20(S), C22(R), and C25(R). Fig.12 It is the molecular structure formula of syringone. Fig.13 and Fig.14 This is the single crystal absolute configuration diagram of serpentine. The crystallographic data of this compound is deposited in the Cambridge Crystallographic Data Center with the number CCDC-2340543.

[0063] The embodiments described above are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. The use of Fusarium oxysporum in the preparation of syringone, characterized in that: The Fusarium oxysporum is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.40905.

2. The use according to claim 1, characterized in that: The Fusarium oxysporum produces syringone by fermentation under the culture condition containing turmeric.

3. The use according to claim 2, characterized in that: The Fusarium oxysporum fungus converts turmeric into yuccaone.

4. The use according to claim 2, characterized in that: The Fusarium oxysporum produces yuccaone through solid-state fermentation, and the culture medium of the solid-state fermentation contains 70 wt % of turmeric and 4 wt % of yeast extract powder.

5. The use according to claim 4, characterized in that: The solid-state fermentation conditions are: fermentation time 9 days, fermentation temperature 32°C.

6. The use according to claim 4, characterized in that: The solid-state fermentation process is stirred and water is replenished; in terms of mass percentage, the water replenishment amount is 60% of the solid-state fermentation material.

7. The use according to any one of claims 1 to 6, characterized in that: The solid-state fermentation product of Fusarium oxysporum is extracted with petroleum ether as solvent, and syringone is obtained through purification.

8. The use according to claim 7, characterized in that: 5 L of petroleum ether and 1 kg of the dried solid-state fermentation product of Fusarium oxysporum are mixed, and syringone is prepared by ultrasonic-assisted extraction; The ultrasonic parameter conditions are: ultrasonic frequency 99 Hz, ultrasonic time 30 min.

9. Use of the syringone prepared by Fusarium oxysporum according to claim 1 in drug development.

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