Fusarium oxysporum and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and relates to a compound synthesized through microbial fermentation catalysis, in particular to fusarium oxysporum and application thereof. The fusarium oxysporum disclosed by the invention has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.40905. The method for preparing the yamogenin by adopting the fusarium oxysporum specifically comprises the following steps: activating the fusarium oxysporum SYfxl23.3, and culturing a first-grade seed; preparing second-level seeds by using the first-level seeds; adding yellow ginger into the secondary seeds, and performing solid-state fermentation to obtain a yamogenin product; and after fermentation is completed, drying a solid-state fermentation product, and then carrying out ultrasonic extraction by adopting petroleum ether to obtain yamogenin. According to the invention, the fusarium oxysporum is applied to preparation of the yamogenin for the first time, and a biotransformation production process of the yamogenin is established. Compared with the prior art, the method has the advantages of being simple in preparation process, high in product purity, environmentally friendly and low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, relates to the synthesis of compounds by microbial fermentation catalysis, and specifically relates to Fusarium oxysporum and application thereof. Background Art

[0002] Depression is a multifaceted psychiatric disorder that manifests itself as physical symptoms such as fatigue, changes in appetite, and disrupted sleep patterns, accompanied by personal dysfunction. The pathophysiology of depression is complex, and the pathogenesis remains unclear. Inflammation has been reported to be an important factor affecting the pathophysiological progression of depression, and studies have shown that patients with depression have elevated levels of proinflammatory cytokines. The inflammatory process can cause apoptosis of neural cells, including neurons and glial cells in the brain, and the activation of apoptotic pathways contributes to depression-related neurobiological changes. Endoplasmic reticulum stress is particularly relevant to depression.

[0003] Yamogenin has a variety of pharmacological properties. Some scholars used lipopolysaccharide (LPS) to induce a mouse depressive-like behavior model and proved that yamogenin enhanced the mouse's sucrose preference and reduced the immobility time in the forced swimming test. By regulating the PERK / eIF2α / ATF4 / CHOP signaling pathway in the prefrontal cortex, the attenuation of endoplasmic reticulum stress can be observed. In addition, yamogenin enhances the expression of the anti-apoptotic protein Bcl-2, thereby inhibiting the expression of the pro-apoptotic protein caspase-3; yamogenin has an inhibitory effect on the activation of microglia, but does not promote brain-derived neurotrophic factor (BDNF) signal transduction. Yamogenin exerts an antidepressant effect in LPS-induced depression-like model mice by inhibiting endoplasmic reticulum stress and activating microglia. In addition, studies have found that yamogenin is closely related to human depression. As a natural antidepressant, yamogenin will provide new treatment ideas for patients with depression. At present, there is no literature report on the use of Fusarium oxysporum to directly ferment turmeric as raw material to obtain yamsapogenin in batches. It is also difficult for ordinary technicians in this field to know whether Fusarium oxysporum can be used to obtain yamsapogenin from turmeric by solid-state fermentation. The inventors of this patent application have conducted in-depth research and explored the application of Fusarium oxysporum to microbial transformation to obtain yamsapogenin, and explored a clean production process with simple steps for the industrial production of yamsapogenin. Summary of the invention

[0004] To overcome the above-mentioned prior art problems, the object of the present invention is to provide a kind of Fusarium oxysporum and its application in the preparation of yam sapogenin. The present invention uses Fusarium oxysporum SYfxl23.3 for microbial transformation to prepare yam sapogenin, and provides a clean bioconversion technology scheme for yam sapogenin. Compared with the prior art, the advantage of the present invention is that Fusarium oxysporum SYfxl23.3 obtained by self-isolation in this laboratory is used to ferment turmeric as raw material to prepare yam sapogenin. The steps for preparing yam sapogenin are simple, the product purity obtained is high, the preparation process has few pollutants, and the market application potential is broad. The applicant of the present invention further studied and clarified the activation culture medium, fermentation culture medium, and fermentation culture method of Fusarium oxysporum SYfxl23.3, and obtained yam sapogenin, which can effectively reduce the production cost of yam sapogenin and develop a clean yam sapogenin bioconversion production process.

[0005] The present invention provides a Fusarium oxysporum SYfx123.3, and the preservation information of the Fusarium oxysporum is as follows:

[0006] Strain name: SYfxl23.3

[0007] Classification name: Fusarium oxysporum

[0008] Date of receipt by the depository: October 27, 2023;

[0009] Time of issuance of preservation certificate: December 27, 2023;

[0010] Depository: China General Microbiology Center (CGMCC);

[0011] Deposit number: CGMCC No.40905.

[0012] The invention relates to an application of Fusarium oxysporum SYfxl23.3, specifically to the application of Fusarium oxysporum SYfxl23.3 to prepare yamgenin through microbial transformation.

[0013] Specifically, Fusarium oxysporum SYfxl23.3 is inoculated on turmeric, and yam sapogenin is obtained through fermentation and biotransformation. The method comprises: inoculating activated Fusarium oxysporum into a primary seed solution, and fermenting and culturing; inoculating the primary seed solution after fermentation and culturing into a secondary seed solution, and fermenting and culturing; adding turmeric, sucrose and beef extract to the secondary seed solution after fermentation and culturing, and fermenting; ultrasonically extracting the fermentation product with an organic solvent, and re-dissolving and drying the extracted product to obtain yam sapogenin.

[0014] Specifically, the components of the primary seed culture medium and the secondary seed culture medium are the same, namely: 5.8g sucrose, 3.0g beef extract, 8.4g Dioscorea rhizome powder, 0.5g K2HPO4, 0.4g KCl, and the volume is adjusted to 1000mL with water.

[0015] Specifically, in terms of mass percentage, the inoculation amount of the primary seed solution is 6% of the secondary seed solution, and the fermentation culture conditions of the primary and secondary seeds are: fermentation time 36h, fermentation speed 150rmp, fermentation temperature 32°C.

[0016] Specifically, in the solid-state fermentation process, the amount of turmeric added is 60% of the secondary seed liquid, and the amount of sucrose and beef extract added is 4% of the secondary seed liquid, by mass percentage.

[0017] Specifically, the turmeric is turmeric powder passed through a 100-mesh sieve, the sucrose is a conventional reagent, and the beef extract is a biological reagent, and the beef extract is produced by Beijing Aoboxing Biotechnology Co., Ltd.

[0018] Specifically, the conditions of the solid-state fermentation are: fermentation time 8 days, fermentation temperature 32° C., stirring and water replenishment during the solid-state fermentation process; in terms of mass percentage, the water replenishment amount is 50% of the solid-state fermentation material.

[0019] Specifically, petroleum ether is selected as the organic solvent, and the solid-state fermentation product is dried and crushed and then ultrasonically assisted extracted with petroleum ether as the solvent; the drying temperature of the solid-state fermentation product is 50°C; the mass volume ratio of the dried solid-state fermentation product to petroleum ether is 1:5; the ultrasonic parameter conditions are: ultrasonic frequency 99 Hz, ultrasonic time 40 min.

[0020] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0021] Compared with the prior art, the present invention applies Fusarium oxysporum to biotransformation, prepares yamogenin by fermentation, and uses turmeric as the main raw material to produce yamogenin. The preparation steps are simple and environmentally friendly, the obtained product has high purity and low production cost, and has excellent industrial development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below 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.

[0023] Figure 1 This is the colony morphology of Fusarium oxysporum strain after being cultured on PDA medium for 2 days.

[0024] Figure 2 This is the bacterial morphology of the Fusarium oxysporum strain after 5 days of fermentation.

[0025] Figure 3 This is a picture of the mycelium morphology of the Fusarium oxysporum strain after staining with lactic acid carbolic acid cotton blue, magnified 100 times.

[0026] Figure 4 This is a morphological image of the hyphae and spores of Fusarium oxysporum strains under a scanning electron microscope.

[0027] Figure 5 This is a diagram of the solid-state fermentation process.

[0028] Figure 6 It is a yamogenin powder product separated and purified from solid-state fermentation products.

[0029] Figure 7 This is the H NMR spectrum of yamgenin.

[0030] Figure 8 This is the carbon NMR spectrum of yamgenin.

[0031] Fig. 9 This is the DEPT spectrum of yamgenin.

[0032] Fig.10 This is the HSQC spectrum of yamgenin.

[0033] Fig.11 This is the high-resolution mass spectrum of yamgenin.

[0034] Fig.12 This is the Fourier transform infrared spectrum of yamgenin.

[0035] Fig.13 It is the molecular structure formula of yamgenin. DETAILED DESCRIPTION

[0036] 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 available on the market unless otherwise specified, and the % in the following examples are all mass percentages unless otherwise specified.

[0037] Example 1

[0038] This example provides a strain of Fusarium oxysporum (F.oxysporum) SY fxl 23.3 Collection and separation.

[0039] Rinse the rhizomes of Dioscorea with water, place them on a clean bench, and cut them into 5cm×5cm small pieces for later use; first, soak the rhizomes of Dioscorea in 75% ethanol for 30s, take them out and rinse them with sterile water for 4 times, then soak them in 0.01% HgCl2 for 3min, take them out and rinse them with sterile water for 4 times; after the above sterilization operation is completed, cut the rhizomes of Dioscorea into a length of about 0.5cm, inoculate them into culture dishes, inoculate 2 pieces in each culture dish, do 3 replicates, and place them in a 28°C incubator for 5d; after colonies grow around the tissue blocks, pick the bacteria with an inoculation loop, streak them on the PDA culture medium, seal and mark them, and then culture them in a 28°C constant temperature incubator; after colonies grow, repeat this step, streak them repeatedly, and after obtaining a single colony, use a PDA slant culture base to store them at 4°C for later use, and isolate the Fusarium oxysporum (F.oxysporum) strain SY fxl 23.3.

[0040] 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 16SrDNA 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 16SrDNA gene sequence of the Fusarium oxysporum strain and the nearest valid strain is 99%.

[0041] 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. Figure 3The 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 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 16S rDNA identification results, the Fusarium oxysporum described in the present invention belongs to the genus Fusarium.

[0042] Example 2

[0043] This embodiment provides a process for preparing the primary and secondary seed solutions of Fusarium oxysporum SYfxl23.3.

[0044] Fusarium oxysporum was inoculated on PDA medium for activation.

[0045] The culture medium was prepared according to the ratio of 5.8g sucrose, 3.0g beef extract, 8.4g Dioscorea rhizome powder, 0.5g K2HPO4, 0.4g KCl and the balance water per liter of culture medium. The prepared culture medium was sterilized by high-pressure wet heat and then cooled to room temperature to obtain a seed culture medium.

[0046] The activated strain was inoculated on the above seed culture medium, and the first-grade seed solution was obtained after shaking flask fermentation for 36 hours (fermentation speed 140rmp, fermentation temperature 32°C).

[0047] Then, the obtained first-level seed liquid was inoculated into the seed culture medium (the culture medium used was the same as the seed culture medium for preparing the first-level seed liquid) according to 6% of the second-level seed liquid, and the second-level seed liquid was obtained after shaking flask fermentation for 24 hours (fermentation speed 140rmp, fermentation temperature 32°C).

[0048] In the above method, the primary seed culture medium and the secondary seed culture medium are the same, the culture process is simpler, and the seeds have strong vitality, which provides a guarantee for the later solid-state fermentation.

[0049] Example 3

[0050] This embodiment provides a process for preparing yamgenin by solid-state fermentation of Fusarium oxysporum.

[0051] Take an appropriate amount of turmeric, crush it and pass it through a 100-mesh sieve to obtain turmeric powder.

[0052] 4% sucrose and beef extract (biological reagent, Beijing Aoboxing Biotechnology Co., Ltd.) of the secondary seed solution were weighed and stirred evenly to obtain a solid fermentation medium.

[0053] The turmeric powder was inoculated into the secondary seed solution of Fusarium oxysporum SYfxl23.3 (see Example 2 for the preparation steps) at 60% of the secondary seed solution, and then the solid fermentation medium was added to carry out solid fermentation at 32°C for 8 days. During the fermentation process, the mixture was fully stirred and 50% of water was supplied. The fermentation process was as follows: Figure 5 shown.

[0054] After the fermentation is completed, the solid fermentation product is placed in an oven at 50°C for drying and then crushed. 4g of the crushed powder is sampled by a uniform method, 20ml of petroleum ether is added and mixed, ultrasonic extraction is performed at 99Hz for 40min, and the mixture is placed in a shaking table with a rotation speed of 150rpm overnight, and 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 yam sapogenin after the collected liquid evaporates.

[0055] Add chloroform to the crude product of yamgenin to dissolve it again, and dilute it to 10 ml in a volumetric flask. After the liquid component in the obtained solution is completely volatilized, yamgenin powder is obtained. Figure 6 shown.

[0056] Compared with the prior art, the present embodiment adopts solid-state fermentation to prepare yam sapogenin from turmeric, the preparation steps are simple, the preparation process is environmentally friendly, the obtained yam sapogenin has low cost, and has good industrial application prospects.

[0057] Example 4

[0058] This example provides the structural identification of yamgenin.

[0059] By performing nuclear magnetic resonance analysis on the product of Example 3, the molecular structure of the product is analyzed and the chemical structure of the product is determined.

[0060] The molecular formula of this compound is C 27 H 42 O3, in 1 H-NMR spectrum Figure 7In the spectral spectrum of the compound, except for one olefinic proton (δH5.33, s) and two tertiary carbon proton signals connected to oxygen (δH4.39, q; 3.46, m) and two secondary carbon atoms connected to oxygen (δH3.38, m), the proton signals of the compound are mainly concentrated in the high field region, and the signals overlap seriously. Four methyl signals can be observed in the high field region, of which two methyls are angular methyls (δH0.79, s, 3H; 0.96, s, 3H), and the other methyl peaks are superimposed multiple peaks (δH0.79, m, 3H) and doublets (δH0.98, d, J = 6.8 Hz, 3H). The hydrogen spectrum of the compound conforms to the typical characteristics of C27 steroid compounds. 13 C-NMR spectrum Figure 8 27 carbon signal peaks belonging to the structure can be observed in the DEPT spectrum. Fig. 9 The 27 carbon atoms are divided into three sp hybridized quaternary carbon atoms (δC 37.08, 40.23, 109.73), one sp 2 Hybridized quaternary carbon (δC141.26), 8 sp hybridized tertiary carbons (δC30.74, 32.05, 42.05, 50.49, 56.96, 62.53, 72.14, 81.26), 1 sp 2 Hybridized tertiary carbon (δC121.85); 10 methylene signals (δC21.31, 29.24, 31.82, 31.88, 32.29, 32.49, 37.67, 40.70, 42.72, 67.28); 4 methyl carbon signals (δC14.97, 16.73, 17.59, 21.31). The compound has two tertiary carbons (δC50.49, 56.96) with chemical shifts around 50ppm, which is in line with the typical characteristics of steroids or tetracyclic triterpenoids. Combined with the fact that the compound has 27 carbons and the hydrogen spectrum is consistent with the characteristics of phytosterols, it is comprehensively judged that the compound is a C27 ​​steroid containing a double bond. Combined with the HSQC two-dimensional spectrum Fig.10 The above-mentioned related information of connected hydrogen and carbon was further confirmed to assist in verifying the correctness of the attribution. The verification results are shown in Table 1. According to the LC-MS spectrum Fig.11 Ms shows that the molecular weight of the substance is 415.32098 (M+1), and the error between the theoretical value and the actual peak value is less than 5*10 -6 . Fourier transform infrared spectrum Fig.12 It shows that the substance exists at 3447cm -1 The peaks near the -OH hydroxyl vibration signal, and 2943 and 2841 cm -1 The peak nearby is the alkyl CH vibration signal, 1602cm -1 The peak nearby is the C=C vibration signal of olefins, 1361 cm -1 The peak near is the bending vibration signal of alkyl CH, 1063cm-1 The peaks near the alkyl CO stretching vibration signals. Comparison of NMR data, mass spectrometry information, and infrared data in the scifinder database confirmed that the compound was Yamogenin, and the molecular formula of the compound was C 27 H 42 O3, molecular structure Fig.13 .

[0061] Table 1 Hydrogen and carbon spectrum data of yam saponin

[0062]

[0063] In this example, nuclear magnetic resonance analysis and mass spectrometry were performed on the yamgenin product, and the identification result showed that the product of Example 3 was yamgenin.

[0064] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred implementation modes of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A Fusarium oxysporum, It is characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.40905.

2. Use of the Fusarium oxysporum according to claim 1 in the preparation of yamgenin.

3. The use according to claim 2, It is characterized in that Fusarium oxysporumSYfxl23.3 was directly inoculated into turmeric, and yam sapogenin was directly obtained through solid-state fermentation and product separation.

4. The use according to claim 3, It is characterized in that The main raw material for solid-state fermentation is turmeric, and the auxiliary raw materials include sucrose and beef paste.

5. The use according to claim 3 or 4, It is characterized in that The components of the primary and secondary seed culture media used in the solid-state fermentation process are the same.

6. The use according to claim 5, It is characterized in that The components of the seed culture medium include: sucrose, beef extract, yam rhizome powder, dipotassium hydrogen phosphate, potassium chloride and water.

7. The use according to claim 3 or 4, It is characterized in that In the solid-state fermentation process, the amount of turmeric added is 60% of the secondary seed liquid by mass percentage.

8. The use according to claim 7, It is characterized in that Stir and add 50% water during solid-state fermentation.

9. The use according to any one of claims 2 to 4, It is characterized in that The purification steps of the fermentation product include drying, crushing, extraction, re-dissolving and drying of the solid fermentation product.

10. The use according to claim 9, It is characterized in that The drying temperature of the solid-state fermentation product is 50°C, the extraction method is ultrasonic extraction, and the volume mass ratio of petroleum ether to the solid-state fermentation product in ultrasonic extraction is 5:1.

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