Mortierella ramanniana strain, medium and use thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a Mattirolomyces spinosus strain with antioxidant activity, a composition containing the strain, and a culture medium for culturing the strain. The preservation number of the Mattirolomyces spinosus strain is CGMCC NO. 42557. The strain and the fermentation culture thereof can eliminate DPPH free radicals, ABTS free radicals and hydroxyl free radicals.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to *Maltese spp.* strains, culture media for culturing *Maltese spp.* strains, and the use of their mycelia. Technical Background

[0002] *Mattirolomyces spinosus* (≡ *Terfezia spinosa*) is a rare underground fungus belonging to the genus *Mattirolomyces* in the family Pezizaceae. *Mattirolomyces spinosus* was first recorded in my country in 1979 in the *Compendium of Fungi of China*, edited by Dai Fanglan. Subsequent reports have primarily relied on catalogues or scattered descriptions, lacking supporting evidence such as specimens and molecular sequences. Furthermore, *Mattirolomyces spinosus*, along with two other species in the genus, *M. terfezioides* (≡ *Terfezia terfezioides*) and *Terfezia arenaria*, is listed in my country's edible macrofungi list, indicating that this genus of fungi possesses certain edible and potential development value.

[0003] However, wild resources of *Aspergillus niger* are scarce and difficult to obtain, and artificial cultivation is also challenging. Liquid fermentation technology, on the other hand, offers advantages such as a wide availability of culture medium raw materials, low cost, short fermentation cycle, high yield, and good controllability, making it a more effective and safer method. During fermentation, the mycelium produces various bioactive components such as polysaccharides, flavonoids, polyphenols, and terpenoids, exhibiting anticancer, anti-inflammatory, and antibacterial effects. Currently, liquid fermentation technology has been applied to the cultivation of various edible fungi, successfully developing products related to edible fungi mycelium and fermentation broth. Therefore, establishing methods for isolating pure culture strains of *Aspergillus niger*, optimizing culture medium formulations, and establishing methods for liquid fermentation of mycelium are of great significance for alleviating the shortage of *Aspergillus niger* resources and promoting the development of bioactive substance products. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a strain of *Maltese spp.* with antioxidant activity, as well as the culture medium, mycelial culture method, and uses of the mycelium of the strain, in order to solve the technical problems of resource shortage of *Maltese spp.* and difficulty in developing active substances, thereby facilitating its subsequent application in health products, cosmetics, or pharmaceuticals.

[0005] The objectives of this invention and the solutions to its technical problems can be achieved by the following technical solutions.

[0006] On one hand, this invention provides a strain of *Mattirolomyces* MS1. This strain was isolated from the ascocarps of *Mattirolomyces* collected in Shangqiu City, Henan Province on October 21, 2025, and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 16, 2026, with the accession number CGMCC No. 42557. The taxonomic name of this strain is *Mattirolomyces*; its Latin scientific name is *Mattirolomyces spinosus*.

[0007] In an embodiment of the first aspect, the rDNA-ITS sequence of *Maltese spp.* strain MS1 ​​is shown in SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides a culture medium for culturing *Mortisus spp.* strain MS1, comprising, based on 1000 mL of deionized water: 0.05 g CaCl2, 0.025 g NaCl, 0.5 g KH2PO4, 0.15 g MgSO4·7H2O, 0.25 g (NH4)2HPO4, 5 g malt extract, 10 g glucose, 0.0001 g vitamin B1, 0.01 g CuSO4, and 15 g agar.

[0009] In the second embodiment, the *Matithia spp.* strain MS1 ​​was cultured in the dark in the culture medium of the second aspect. After 5 days, the hyphae of MS1 began to germinate. The hyphae were white and relatively sparse. After 10 days, the colonies were round or nearly round, flat, and expanded radially to the periphery. The hyphae were evenly distributed and the edges were continuous but irregular. After 20 days, the colonies further expanded, and the hyphae were evenly distributed and intertwined to form a distinct villous structure.

[0010] In a third aspect, the present invention provides an antioxidant, which is the above-mentioned *Maltese cauliflorus* strain MS1 ​​or a fermentation culture of *Maltese cauliflorus* strain MS1.

[0011] In a fourth aspect, the present invention provides a composition for preventing and / or treating oxidative damage, the composition comprising mycelium of *Mortisus caudatus* strain MS1 ​​or a fermentation culture of *Mortisus caudatus* strain MS1.

[0012] In a fifth aspect, the present invention provides the use of *Mortisus caudatus* strain MS1 ​​mycelium or *Mortisus caudatus* strain MS1 ​​fermentation culture in the manufacture of pharmaceutical compositions for the prevention and / or treatment of oxidative damage.

[0013] This invention offers significant advantages over existing technologies. It provides a *St. caudatus* strain MS1 ​​with antioxidant activity. The mycelium of this strain can scavenge DPPH free radicals, ABTS free radicals, and hydroxyl free radicals, exhibiting antioxidant activity and thus providing a composition for preventing and / or treating oxidative damage. The culture medium for *St. caudatus* strain MS1 ​​provided by this invention allows the strain to grow at a faster rate, which helps address the problems of *St. caudatus* resource shortage and difficulties in developing active substance products. Furthermore, the mycelium described in this invention has the potential to act as a natural antioxidant and can be used in the development and application of food, health food, cosmetics, and pharmaceutical-related products.

[0014] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0015] Figure 1 The image shows *Matitis repens* (A) and the isolated strain (B).

[0016] Figure 2 The antioxidant activity of water-soluble crude polysaccharides from *Maltis cristatus* mycelium and ascocarp is compared. A: ABTS radical scavenging rate; B: DPPH radical scavenging rate; C: hydroxyl radical scavenging rate.

[0017] The strain MS1 ​​of *Mattirolomyces spinosus* has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No: 42557 and deposit date of January 16, 2026. The taxonomic name of this strain is *Mattirolomyces spinosus*. Detailed Implementation

[0018] The preferred embodiments of the present invention will now be described in detail with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent, and all such modifications and substitutions fall within the scope of protection claimed in the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0020] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2012), or as recommended by the manufacturer’s instructions.

[0021] Unless otherwise specified or obvious from the context, the term "about" as used herein shall be understood to mean within the normal tolerance range in the art, such as within two standard deviations of the average. "About" may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless the context clearly indicates otherwise, all numerical values ​​provided in this specification and claims may be modified by the term "about".

[0022] Example 1: Isolation of pure culture strains of *Rhizoctonia solani*

[0023] 1.1 Culture characteristics

[0024] Prepare a solid culture medium using the following ingredients: 0.05 g CaCl2, 0.025 g NaCl, 0.5 g KH2PO4, 0.15 g MgSO4·7H2O, 0.25 g (NH4)2HPO4, 10 g glucose, 15 g agar, and 1000 mL deionized water. Sterilize the medium at 121°C for 30 min, then cool it to 50-60°C. In a clean bench, dispense 15-20 mL of the medium into 90 mm diameter disposable sterile plastic petri dishes and allow them to cool and solidify for later use.

[0025] Fresh, tuberous ascocarps of the fungus were collected from Shangqiu, Henan Province on October 21, 2025. Figure 1 A) Remove external mud and sand, prioritizing ascocarps with dense tissue, free from rot and insect infestation, and process them under sterile conditions. Rinse the outer surface of the ascocarp sequentially with sterile water; treat with 75% ethanol for 60 seconds; then treat with 5% sodium hypochlorite disinfectant for 2 minutes; subsequently rinse three times with sterile water and dry the surface. In a laminar flow hood, manually break open the ascocarp and use a sterile scalpel to remove a 2-5 mm section of relatively uncontaminated internal tissue. 2Tissue blocks, 1-3 mm thick, were inoculated into the center of a petri dish and incubated at 25±1℃ in the dark. After 5 days, mycelia began to germinate; the mycelia were white and relatively sparse. After 10 days, colonies were round or nearly round, flat, and expanded radially towards the periphery, with evenly distributed hyphae and continuous but irregular edges. After 20 days, the colonies further enlarged, with evenly distributed and interwoven hyphae forming a distinct villous structure. Figure 1 B).

[0026] Using a 5 mm diameter punch, mycelial cakes were collected from the edge of the plate and transferred to a new culture medium. The purification process was repeated 2-3 times to obtain a pure culture strain with consistent morphology, which was named MS1.

[0027] 1.2 Identification by internal transcribed spacer (ITS) sequence

[0028] Mycelial DNA was extracted from strain MS1 ​​using the CTAB method. Using the DNA as a template, PCR amplification was performed using universal primers ITS1 / ITS4 (see Li Jiamei, Liu Peigui, Qiao Peng, et al. Distribution and ecological significance of Indian Truffle in Northeast my country [J]. Journal of Edible Fungi, 2019, 26(2), 97-105). The amplified product was sent to Genewiz (Beijing) Biotechnology Co., Ltd. for sequencing. The sequence of the amplified product is shown in SEQ ID NO: 1.

[0029] The sequencing results (SEQ ID NO: 1) were subjected to Blast similarity analysis in the GenBank nucleic acid database. After Blast sequence alignment and phylogenetic analysis, strain MS1 ​​was identified as Mattirolomyces spinosus.

[0030] Example 2: Optimization of Solid Culture Medium Formulation

[0031] Based on the above solid culture medium, the radial growth rate of *Maltese spiculatus* mycelium under different culture medium conditions was compared by adjusting the concentrations of carbon source, compound nutrient and nitrogen source, in order to optimize the culture medium formulation.

[0032] The recipe is as follows:

[0033] G1 control group: CaCl2 0.05 g, NaCl 0.025 g, KH2PO4 0.5 g, MgSO4·7H2O 0.15 g, (NH4)2HPO4 0.25 g, glucose 10 g, agar 15 g, deionized water 1000 mL, pH adjusted to 6.0-6.5.

[0034] G2: Based on G1, add vitamin B1 0.1 mg / L, otherwise the same as G1;

[0035] G3: Add 5 g / L of malt extract powder to G1, otherwise the same as G1;

[0036] G4: Add 0.01 g / L CuSO4 to G1, otherwise the same as G1;

[0037] G5: Based on G1, add vitamin B1 0.1 mg / L, malt extract powder 5 g / L, and CuSO4 0.01 g / L, the rest are the same as G1.

[0038] After autoclaving, the culture media for each group were quantitatively poured into Petri dishes, and the purified mycelia from Example 1 were quantitatively inoculated. Every two days, colonies were streaked at the bottom of the Petri dishes, and the diameter of the colonies was measured using the "cross-hatching method." The growth rates of the strains are shown in Table 1 below. One-way ANOVA was performed on the mycelial growth rates, and the results (Table 1) showed that the mycelial growth rate of G5 was significantly higher than that of G1 (P<0.01), and also superior to other culture media.

[0039] Table 1. Growth rates of *Maltese spp.* in different culture media

[0040] Group Mycelial growth rate (mm / d) G1 1.94±0.06 G2 2.27±0.12 G3 2.72±0.09 G4 2.15±0.07 G5 2.91±0.05

[0041] Example 3: Preparation of mycelium by liquid fermentation

[0042] Take the pure culture strain obtained in Example 1, cut off the mycelial cake (5 mm in diameter), inoculate it into an Erlenmeyer flask containing liquid culture medium, break it up with an inoculation needle at the flask wall, and culture it at 25°C and 200 rpm for 20 days to obtain the fermentation broth of Strychnos nucifera.

[0043] The liquid culture medium comprises the following components in the following mass concentrations: CaCl2 0.05 g, NaCl 0.025 g, KH2PO4 0.5 g, MgSO4·7H2O 0.15 g, (NH4)2HPO4 0.25 g, malt extract powder 5 g, glucose 10 g, vitamin B1 0.0001 g, CuSO4 0.01 g, and deionized water 1000 mL.

[0044] The fermentation broth of *Rhizoctonia solani* was filtered to obtain filter residue. The filter residue was washed with distilled water, centrifuged, dried at 40-60℃ to constant weight, pulverized and sieved to obtain *Rhizoctonia solani* mycelium.

[0045] Example 4: Preparation and evaluation of antioxidant products from mycelium

[0046] 4.1 Preparation of water-soluble crude polysaccharides from mycelium and ascocarp

[0047] The sieved mycelium and ascocarps of *Mallotus spp.* were extracted with distilled water at a ratio of 1:50 (g: mL) and the mixture was extracted at 90℃ for 2 h. The extraction was repeated three times. The filtrates were combined, concentrated, and then precipitated with 4 times the volume of anhydrous ethanol for 12 h. After centrifugation, the precipitate was redissolved and freeze-dried to obtain the water-soluble crude polysaccharides of *Mallotus spp.* mycelium and ascocarps.

[0048] 4.2 Evaluation of the antioxidant activity of water-soluble crude polysaccharides from mycelium and ascocarp

[0049] The obtained crude polysaccharides were prepared into different concentration gradients and evaluated using methods such as DPPH free radical scavenging, ABTS free radical scavenging, and hydroxyl free radical scavenging. The results showed that both the mycelial extract and the ascocarp extract exhibited antioxidant activity.

[0050] DPPH free radical scavenging ability

[0051] Following the method of Brand-Williams et al. (Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity [J]. LWT-FoodScience and Technology, 1995, 28(1), 25-30), a 0.2 mmol / L DPPH ethanol solution was prepared. Polysaccharide solutions of 1, 2, 3, 4, and 5 mg / mL were prepared. Blank group A0 (2.0 mL anhydrous ethanol + 2.0 mL 0.2 mmol / L DPPH solution), sample group A1 (2.0 mL polysaccharide solution + 2.0 mL 0.2 mmol / L DPPH solution), and control group A2 (2.0 mL polysaccharide solution + 2.0 mL anhydrous ethanol) were set up. After thorough mixing, the mixture was reacted at 25℃ in the dark for 30 min. After centrifugation, the supernatant was measured at 517 nm. L-ascorbic acid was used as a positive control, and three replicates were set up, with the average value taken. Calculation formula: DPPH free radical scavenging rate (%) = [1 – (A1 – A2) / A0] × 100, where: A0 is the absorbance of the reaction system with anhydrous ethanol replacing the sample, A1 is the absorbance of the reaction system with the sample added, and A2 is the absorbance of the reaction system with anhydrous ethanol replacing DPPH.

[0052] ABTS free radical scavenging ability

[0053] The method was followed, as described by Miller et al. (Miller NJ, Rice-Evans C, Davies MJ et al. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates [J]. Clinical Science, 1993, 84(4), 407-412). The ABTS stock solution was prepared by mixing a 2.45 mmol / L potassium persulfate aqueous solution and a 7.0 mmol / L ABTS solution, and then incubating at 25°C in the dark for 12 h. The ABTS working solution was prepared by diluting the ABTS stock solution with distilled water until its absorbance at 734 nm was approximately 0.7. Polysaccharide solutions of 1, 2, 3, 4, and 5 mg / mL were prepared. The following groups were set up: blank group A0 (2.0 mL distilled water + 2.0 mL ABTS working solution), sample group A1 (2.0 mL polysaccharide solution + 2.0 mL ABTS working solution), and control group A2 (2.0 mL polysaccharide solution + 2.0 mL distilled water). After thorough mixing and reaction in the dark at 25℃ for 20 min, the absorbance at 734 nm was measured. L-ascorbic acid was used as a positive control. Three replicates were set up, and the average value was taken. The calculation formula was: ABTS free radical scavenging rate (%) = [1 – (A1 – A2) / A0] × 100, where: A0 is the absorbance of the reaction system with distilled water instead of the sample, A1 is the absorbance of the reaction system with the sample added, and A2 is the absorbance of the reaction system with distilled water instead of ABTS.

[0054] Hydroxyl radical scavenging ability

[0055] Following the method of Xiao et al. (Xiao ZJ, Yan CY, Jia CX, et al. Structural characterization of chia seed polysaccharides and evaluation of its immunomodulatory and antioxidant activities [J]. Food Chemistry: X, 2023, 20,101011), 200 μL of 2.25 mmol / L FeSO4 aqueous solution, 200 μL of 9 mmol / L salicylic acid ethanol solution, 200 μL of a series of concentrations of mycelial water-soluble polysaccharides and ascocarp water-soluble polysaccharide solutions (1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL), and 200 μL of 8.8 mmol / L H2O2 solution were added to centrifuge tubes. The reaction was carried out at 37℃ for 30 min, and the absorbance was measured at 510 nm using a microplate reader. L-ascorbic acid was used as a positive control, and three replicates were set up, with the average value taken. Hydroxyl radical scavenging rate (%) = [1–(A1–A2) / A0]×100, where: A0 is the absorbance of the reaction system with distilled water instead of sample solution, A1 is the absorbance of the reaction system with sample solution added, and A2 is the absorbance of the reaction system with distilled water instead of FeSO4 solution.

[0056] The results are as follows Figure 2 As shown, the water-soluble polysaccharide from *Maltis cristatus* mycelium exhibits antioxidant activity. At 5 mg / mL, the scavenging rates of ABTS and DPPH free radicals by the mycelium water-soluble polysaccharide were 63.64% and 64.63%, respectively. Compared with the water-soluble polysaccharide from *Ascomycota* (46.10% and 37.40%), the scavenging rates were increased by 38.05% and 72.81%, respectively. The scavenging rate of hydroxyl radicals by the mycelium water-soluble polysaccharide was relatively low, only 40.96%, lower than the 67.10% of the *Ascomycota* water-soluble polysaccharide. In conclusion, the mycelium water-soluble polysaccharide possesses certain in vitro antioxidant activity, and its scavenging activity against DPPH, ABTS, and hydroxyl radicals continuously increases with increasing concentration.

Claims

1. Strychnos nucifera ( Mattirolomyces spinosus strain MS1, characterized in that, The strain has the preservation number CGMCC No. 42557.

2. The application of the *Maltese cauliflower* strain MS1 ​​according to claim 1 in the preparation of antioxidants, characterized in that, The antioxidant comprises water-soluble crude polysaccharide from the mycelium of *Maltis cristatus* strain MS1 ​​or water-soluble crude polysaccharide from the ascocarp. The water-soluble crude polysaccharide is prepared by the following method: take the pulverized and sieved mycelium or ascocarp of *Maltis cristatus* strain MS1, add distilled water at a material-to-liquid ratio of 1:50 (g: mL), extract at 90℃ for 2 h, repeat three times, combine the filtrates, concentrate, add 4 times the volume of anhydrous ethanol for alcohol precipitation for 12 h, centrifuge, redissolve the precipitate, freeze-dry to obtain the water-soluble crude polysaccharide from the mycelium of *Maltis cristatus* strain MS1 ​​or water-soluble crude polysaccharide from the ascocarp.

Images