A strain of Phaeacremonium scolyti and its application

Abstract

The application discloses a Phaeoacremonium scolyti strain and application thereof. A laccar symbiotic bacterium is screened from laccar, and is identified as a strain of the Phaeoacremonium genus through morphology and molecular biology, and is named as Phaeoacremonium scolyti CX-2. The bacterium can utilize laccar powder after laccar is combined to carry out fermentation, and active substances are produced, and with the increase of the fermentation time, the laccar alcohol extract content, the laccar characteristic substance content and the laccar alcohol extraction antioxidant activity are significantly improved, so that the bacterium can produce sesquiterpenes and chromones by taking wood as raw material, and has a wide application prospect in the industrial production of laccar.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a strain of Phaeoacremonium scolyti and its applications. Background Technology

[0002] my country boasts a long and rich tradition of incense culture, with agarwood holding a prominent position, often referred to as the "King of Fragrances," possessing a profound historical and cultural significance. The chemical components of agarwood primarily include sesquiterpenes, aromatic compounds, chromones, and fatty acids. These components not only give agarwood its unique aroma but also possess a wide range of pharmacological effects, such as antibacterial, anti-inflammatory, sedative, analgesic, and blood pressure-lowering properties. With the booming domestic economy and the revival of traditional culture, the demand for incense is surging, and agarwood culture is gaining popularity. Its applications in medicine, fragrances, and beauty products are becoming increasingly widespread, leading to a growing demand. However, agarwood production is limited, and high-quality agarwood often requires several years to form. Therefore, improving the aroma quality of agarwood through biotechnology has significant practical and economic value. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Phaeoacremonium scolyti.

[0004] Another object of the present invention is to provide the application of the above-mentioned Phaeoacremonium scolyti bacterium.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A strain of Phaeoacremonium scolyti, obtained from an agarwood sample, was selected and enriched, and named Phaeoacremonium scolyti CX-2.

[0007] The Phaeoacremonium scolyti strain, with accession number GDMCC NO.65425, was deposited on November 6, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The colonies of Phaeoacremonium scolyti on PDA plates are small, reddish-brown, nearly round, with white edges. The hyphae are divided into several segments by septa, each segment containing cytoplasm and one or more nuclei. The spores are numerous and elliptical.

[0009] The Phaeoacremonium scolyti bacteria can ferment agarwood to obtain sesquiterpenes and chromones.

[0010] The application of Phaeoacremonium scolyti in the fermentation and aroma enhancement of agarwood.

[0011] The application of Phaeoacremonium scolyti in the production of sesquiterpenes and chromones.

[0012] The chromones mentioned include at least one of 2-(2-phenylethyl)chromone, 6-methoxy-2-(2-phenylethyl)chromone, 2-(4-methoxyphenylethyl)chromone, and 6,7-dimethoxy-2-(phenylethyl)chromone.

[0013] The sesquiterpenoids mentioned include at least one of β-santalol, azulene, aristolochic acid, (-)-guaiacol, eucalyptol, eucalyptol, β-rutoxene, gemmaconone, cypermethrin, longleaf aldehyde, β-eucalyptol, eucalyptol, dehydrofuranone, caryophyllene aldehyde, irimofenone, isospathulenol, hydroxyvaleric acid, eucalyptol, eucalyptol, eucalyptol oxide, mosdone, epoxy aristolochic acid, alpha-cyperone, turmeric alcohol, eucalyptol lactone, (-)-rohanbane, vetiselinenol, 6,9-guaiacol, 4(15),5,10(14)-geranyltrien-1-ol, draconone, trans-valeric acid ester, ylangenol, and (-)-isolongleaf alcohol.

[0014] A method for enhancing the aroma of agarwood through fermentation includes the following steps:

[0015] Phaeoacremonium scolyti was inoculated into a culture medium to obtain a fermentation broth, which was then sprayed onto agarwood powder for fermentation, yielding a fermentation product rich in sesquiterpenes and chromones.

[0016] The agarwood mentioned is the resinous wood of Aquilaria sinensis, a plant of the Thymelaeaceae family; preferably, the agarwood is agarwood that has undergone resin formation.

[0017] The agarwood powder mentioned is powder that has been pulverized and passed through a 30-50 mesh sieve.

[0018] The resin formation mentioned is either naturally occurring or artificially formed.

[0019] The resinous material is formed for more than one year; preferably, it has been formed for more than one year and aged for more than one year.

[0020] The culture medium is PDB liquid culture medium.

[0021] The cultivation conditions are such that the OD600 is greater than 2.

[0022] The ratio of the fermentation broth to the wood powder is 1-2 mL: 1-2 g.

[0023] The fermentation conditions are as follows: cultured at 26–30°C for at least 3 days.

[0024] The present invention has the following advantages and effects compared with the prior art:

[0025] This invention screened a symbiotic fungus from agarwood, which, after morphological and molecular biological identification, was determined to be a strain of the genus *Phaeoacremonium*, and named *Phaeoacremonium scolyti* CX-2. This fungus can ferment agarwood powder after resin formation to produce active substances. Furthermore, with increasing fermentation time, the content of agarwood alcohol extract, the content of agarwood characteristic substances, and the antioxidant activity of the agarwood alcohol extract all significantly increased, demonstrating that this fungus can produce sesquiterpenes and chromones from wood, and has broad application prospects in the industrial production of agarwood. Attached Figure Description

[0026] Figure 1 Morphology of Phaeoacremonium scolyti CX-2 in culture medium; left image: front view; right image: back view.

[0027] Figure 2 The images show the morphology of spores and hyphae of Phaeoacremonium scolyti CX-2 under a microscope; top image: spore morphology; bottom image: hyphae morphology.

[0028] Figure 3 Phylogenetic tree of Phaeoacremonium scolyti CX-2.

[0029] Figure 4 Photos of raw agarwood; Left: Uncrushed agarwood; Right: Crushed agarwood.

[0030] Figure 5 The figure shows the experimental results of the antioxidant activity of DPPH from agarwood alcohol extract. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0032] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0033] Example 1: Isolation, purification, and identification of Phaeoacremonium scolyti

[0034] Fresh agarwood samples (ensuring they are free from pests and diseases) collected from healthy Aquilaria sinensis trees in the Dongguan Xiangbo Garden were air-dried for one year and then cut into small cubes with sides of approximately 1 cm. After surface sterilization, the samples were cut open and their interiors were placed tightly against PDA plates. The plates were then sealed and placed upright in a (28±1)℃ constant temperature incubator for cultivation. Once the culture medium was fully colonized with mycelium, the strain was purified using the mycelial tip inoculation method. This process was repeated 3–4 times until a pure culture strain was obtained. Finally, one strain that was easy to cultivate and had stable passage characteristics was selected as the target strain, designated CX-2.

[0035] The selected target strain CX-2 was incubated upside down on a PDA plate at (28±1)℃ for 7 days, and the colony morphology was observed. Figure 1 ) Observe the morphology of spores and hyphae under a microscope after taking a bacterial suspension. Figure 2 The colonies of strain CX-2 are small, reddish-brown, nearly circular with white edges. The hyphae are divided into segments by septa, each containing cytoplasm and one or more nuclei. The spores are numerous and elliptical. Based on the *Handbook of Fungal Identification*, CX-2 was initially identified as belonging to the order Sphaeriales. To further determine its classification, molecular biological methods were used to sequence its 18S rDNA. The primers and reaction conditions used are shown in Tables 1-2.

[0036] Table 1 Primer Information

[0037]

[0038]

[0039] Table 2. PCR amplification reaction system and conditions

[0040]

[0041] 18S rDNA nucleotide sequence analysis revealed the strain's 18S rDNA nucleotide sequence to be 599 bp (SEQ ID No. 1). The 18S rDNA nucleotide sequence of the target strain was assembled and compared with the 18S rDNA nucleotide sequences of known bacterial species in the Gen Bank database using the BLAST program on the NCBI website. A phylogenetic tree was then constructed using Mega7 software. The comparison of the strain's 18S rDNA nucleotide sequence with known bacterial species in the Gen Bank database using the BLAST program on the NCBI website showed that this strain was most closely related to *Phaeoacremonium scolyti*, with a gene homology of 99.50%. A phylogenetic tree was constructed using Mega7 software and data from the NCBI database. Figure 1 The results showed that the bacterium was most closely related to Phaeoacremonium scolti DUCC405 (KC013300.1), with a score of 39.

[0042] Based on the above identification results of strain CX-2, CX-2 has been identified as a novel strain of *Phaeoacremonium scolyti*, and named *Phaeoacremonium scolyti* CX-2. This strain was deposited on November 6, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCCNO.65425. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0043] Example 2: Agarwood Fermentation and Aroma Enhancement Experiment

[0044] (1) Take 1 ml of purified Phaeoacremonium scolyti CX-2 bacterial culture and inoculate it into 100 ml of PDB liquid medium. Incubate at (28±1)℃ and 120 rpm for about 2 days until the OD600 reaches 2.0. Then, centrifuge at 4000 rpm for 5 min, remove the supernatant, add 250 ml of sterile water, and shake for 2 min to obtain the fermentation broth.

[0045] (2) Take resinous wood from Aquilaria sinensis (purchased from Dongguan Xiangcheng Industrial Co., Ltd.), which produces resin in one year. After harvesting, age it for one year, crush it, and pass it through a 40-mesh sieve. Weigh 100g of agarwood powder and spray the fermentation liquid obtained in step (1) into the agarwood powder at a ratio of 2:1 (g:ml). Place the inoculated agarwood powder in a constant temperature incubator at (28±1)℃ for 3 days.

[0046] Example 3

[0047] The experiment was conducted according to the method of Example 2, except that the fermentation time was 4 days.

[0048] Example 4

[0049] The experiment was conducted according to the method of Example 2, except that the fermentation time was 5 days.

[0050] Comparative Example 1

[0051] The experiment was conducted according to the method of Example 2, except that the fermentation broth was replaced with the same volume of sterile water.

[0052] Example 5

[0053] After fermentation, the agarwood was placed in a 45℃ oven for 24 hours. The determination of its alcohol-soluble extract content followed the cold maceration method in the Pharmacopoeia of the People's Republic of China (2015 edition): Approximately 4g of the fermented and dried agarwood sample was accurately weighed and placed in a 250ml Erlenmeyer flask. 100ml of 95% ethanol was accurately added, the flask was sealed, and the sample was cold-macerated. The flask was shaken frequently for the first 6 hours, then allowed to stand for 18 hours. The sample was then rapidly filtered through a drying filter. 20ml of the filtrate was accurately measured and placed in an evaporating dish that had been dried to constant weight. The sample was evaporated to dryness on a water bath, then dried at 105℃ for 3 hours. After cooling in a desiccator for 30 minutes, the sample was rapidly and accurately weighed. The content (%) of alcohol-soluble extract in the sample was calculated based on the dried product.

[0054] Alcohol extract content:

[0055]

[0056] Note: χ – Alcohol extract content (%)

[0057] m1 — Mass of the evaporating dish (g);

[0058] m2 — Total mass (g) of the evaporating dish and sample;

[0059] M — Sample mass (g).

[0060] The test results are shown in Table 3:

[0061] Table 3. Content of Agaric Alcohol Extract

[0062] Example Alcohol extract content (%) Unfermented 3.74 Example 2 4.04 Example 3 4.28 Example 4 4.80 Comparative Example 1 3.81

[0063] Experimental results showed that the alcohol extract content in Examples 2-4, after bacterial fermentation, was significantly increased compared to the unfermented agarwood, proving that the bacteria can increase the alcohol extract content of agarwood. Furthermore, within a certain fermentation time, the increase in alcohol extract content increased with increasing fermentation time. Compared to Comparative Example 1, the fermentation of *Phaeoacremonium scolyti* broth increased the alcohol extract content of agarwood by 1%–20%; compared to unfermented agarwood, the fermentation of *Phaeoacremonium scolyti* broth increased the alcohol extract content by 8.02%–28.34%. The comparison between the comparative example and the unfermented agarwood indicates that adding sterile water for fermentation did not significantly increase the alcohol extract content.

[0064] Example 6

[0065] The contents of sesquiterpenes, chromones, aromatic compounds, etc. in the agarwood alcohol extract obtained in Example 5 were detected by gas chromatography-mass spectrometry (GC-MS).

[0066] GC-MS sample preparation:

[0067] Dissolve the alcohol extract in 25 ml of anhydrous ethanol, filter through a 0.45 μm microporous membrane, and prepare the GC-MS sample.

[0068] GC-MS detection conditions:

[0069] a) Chromatographic column: HP-5 (30m × 0.25mm × 0.3μm);

[0070] b) Column temperature: linear program temperature increase from 50℃ to 140℃ at a rate of 3℃ / min; then linear program temperature increase from 140℃ to 160℃ at a rate of 1℃ / min; then linear program temperature increase from 160℃ to 280℃ at a rate of 4℃ / min, hold for 10 minutes.

[0071] c) Carrier gas: Nitrogen, purity ≥ 99.999%, flow rate 1.0 mL / min;

[0072] d) Inlet temperature: 250℃;

[0073] e) Detector temperature: 280℃;

[0074] f) Detector: Hydrogen flame ionization detector;

[0075] g) Injection volume: 1.0 μL;

[0076] h) Flow split ratio: 10:1;

[0077] i) Ion source temperature 230℃;

[0078] j) Ionization energy 70 eV;

[0079] k) Full scan mode;

[0080] l) Scanning range 50~500m / z.

[0081] The test results are shown in Table 4:

[0082] Table 4. Component Analysis of Agaric Alcohol Extract

[0083]

[0084]

[0085]

[0086]

[0087] Note: "-" indicates that the substance was not detected; chromones include 2-(2-phenylethyl)chromone, 6-methoxy-2-(2-phenylethyl)chromone, 2-(4-methoxyphenylethyl)chromone, and 6,7-dimethoxy-2-(phenylethyl)chromone; sesquiterpenes include β-santalol, azulene, aristolochne, (-)-guaiacol, eucalyptol, eucalyptol, β-rutoxene, geraniol, cephalotaxelin, cephalotaxelin, cephalotaxelin, β-eucalyptol, eucalyptol, dehydrofuranone, and carnation. Enal, irimofenone, Isospathulenol, hydroxyvaleric acid, eucalyptol, eucalyptol, eucalyptol oxide, mosdone, epoxyargentinene, alpha-cyperone, turmeric alcohol, eucalyptol lactone, (-)-rohanbiumene, vetiselinenol, 6,9-guaiacdiene, 4(15),5,10(14)-geranyltrien-1-ol, draconinone, trans-valeric acid ester, Ylangenol, (-)-isolongol.

[0088] Experimental results showed that with increasing fermentation time, the total content of terpenoids and chromones in agarwood in Examples 2-4 generally showed an increasing trend. As can be seen from Example 4 in Table 4, the fermentation broth of *Phaeoacremonium scolyti* significantly increased the content of characteristic substances in agarwood oil. The total content of sesquiterpenoids in the fermented agarwood oil was 2.86 mg / ml, and the total content of chromones was 3.28 mg / ml. Compared to Comparative Example 1, the total content of sesquiterpenoids increased by 27.11%, and the total content of chromones increased by 13.49%. Compared to unfermented agarwood, the total content of sesquiterpenoids increased by 31.80%, and the total content of chromones increased by 13.10%. However, compared to unfermented agarwood, the total content of sesquiterpenoids and chromones in the agarwood alcohol extract of Comparative Example 1 did not show significant changes.

[0089] Example 7: Determination of DPPH antioxidant activity of agarwood alcohol extract

[0090] A mixture of 100 μL each of agarwood alcohol extract and DPPH solution was used as the experimental group, with ethanol as the blank control. After standing in the dark for 30 min, the absorbance was measured at 517 nm. The clearance rate (%) was calculated using the formula: Clearance Rate (%) = (A0 - A S ) / A0×100% (where A0 is the absorbance of the blank control, A S The clearance rate is calculated based on the absorbance of the sample to be tested.

[0091] Experimental results are as follows Figure 5 As shown, it can be seen that the antioxidant capacity of DPPH of agarwood alcohol extract is stronger with increasing fermentation time. Compared with unfermented, the antioxidant capacity of the examples increased by 1.01% to 2.41%; compared with Comparative Example 1, the antioxidant capacity of the examples increased by 0.31% to 1.38%.

[0092] In summary, the Phaeoacremonium scolyti CX-2 bacterium in this invention can ferment wood powder from Thymelaeaceae plants to produce active substances. Furthermore, with increasing fermentation time, the content of agarwood alcohol extract, the content of agarwood characteristic substances, and the antioxidant activity of agarwood alcohol extract are significantly improved, proving that this bacterium can produce sesquiterpenes and chromones from wood as raw materials, and has broad application prospects in the industrial production of agarwood.

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of *Phaeoacremonium scolyti*, characterized by: The sample, named Phaeoacremonium scolyti CX-2, was obtained from an agarwood sample through screening and enrichment. Its accession number is GDMCC NO.65425. It was deposited on November 6, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of Phaeoacremonium scolyti as described in claim 1 in the fermentation and aroma enhancement of Aquilaria sinensis.

3. The application of Phaeoacremonium scolyti as described in claim 1 in the production of sesquiterpenes and chromones by fermentation of Aquilaria sinensis.

4. A method for enhancing the aroma of agarwood through fermentation, characterized in that... Includes the following steps: The Phaeoacremonium scolyti strain described in claim 1 was inoculated into a culture medium, and a fermentation broth was obtained. The broth was then sprayed onto agarwood powder and fermented to obtain a fermentation product rich in sesquiterpenes and chromones. The agarwood mentioned is the resinous wood of the Aquilaria sinensis plant (family Thymelaeaceae). The agarwood powder mentioned is powder that has been pulverized and passed through a 30-50 mesh sieve; The culture medium is PDB liquid culture medium; The cultivation conditions are as follows: the culture is carried out until the OD600 is greater than 2; The ratio of the fermentation broth to the wood powder is 1-2 mL: 1-2 g; The fermentation conditions are as follows: cultured at 26–30°C for at least 3 days.

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