Phaeosaccharomonas scolyti bacterium and application thereof

Abstract

The invention discloses a strain of Phaeosaccharomonas scolyti and an application of the strain of Phaeosaccharomonas scolyti. According to the invention, an agilawood symbiotic bacterium is obtained by screening from agilawood, and the agilawood symbiotic bacterium is identified as a strain of Phaeosaccharomonas through morphology and molecular biology, and is named as Phaeosaccharomonas scolyti CX-2. The strain can be fermented by utilizing agilawood powder after agilawood formation to produce active substances, and along with the increase of fermentation time, the content of agilawood alcohol extract, the content of agilawood characteristic substances and the antioxidant activity of agilawood alcohol extraction are remarkably improved, so that the strain is proved to be capable of producing sesquiterpene and chromone by taking wood as a raw material; the method has a wide application prospect in the industrial production of agilawood.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a strain of Phaeoacremonium scolyti and application thereof. Background Art

[0002] my country has a long tradition of incense culture, among which agarwood ranks first and can be called the king of all incense, with a long history and cultural heritage. The chemical components of agarwood mainly include sesquiterpenes, aromatics, chromones and fatty acids. These components not only give agarwood a unique aroma, but also have a wide range of pharmacological effects, such as antibacterial, anti-inflammatory, sedative, analgesic, and blood pressure lowering. With the prosperity of the domestic economy and the revival of traditional culture, the demand for incense has become stronger, and the agarwood culture has become popular. The application of agarwood in many fields such as medicine, spices, and beauty products has become increasingly widespread, and the demand for agarwood has become more and more vigorous. However, the output of agarwood is limited, and high-quality agarwood often takes several years to form. Therefore, how to improve the aroma quality of agarwood through biotechnology has important practical significance and economic value. Summary of the invention

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

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

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A strain of Phaeoacremonium scolyti, derived from agarwood samples, was obtained through screening and enrichment and was named Phaeoacremonium scolyti CX-2.

[0007] The Phaeoacremonium scolyti bacterium has a deposit number of GDMCC NO.65425, and was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on November 6, 2024. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The Phaeoacremonium scolyti bacteria has a small colony on the PDA plate, is reddish brown, nearly circular, and has white edges. The hyphae are divided into several sections by diaphragms, each of which contains cytoplasm and one or more nuclei. The spores are numerous and oval.

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

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

[0011] The Phaeoacremonium scolyti bacterium is used in producing sesquiterpenes and chromone substances.

[0012] The chromone substances 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 sesquiterpenoid substances include at least one of β-santalol, azurene, aristolochene, (-)-guaiacol, eucalyptus globulus alcohol, costus alcohol, β-humulene, germacron, cedarwooddiol, longifole aldehyde, β-eudesmol, eucalyptus spathulone, dehydrofuranone, caryophyllene aldehyde, erimofenone, isospathulenol, hydroxyvaleric acid, eudesmol, eucalyptus oxide, mosdone, epoxy aristolochene, alpha-cyperone, curcumol, inulinolactone, (-)-roupene, vetiselinenol, 6,9-guaiadiene, 4(15),5,10(14)-germinol-1-ol, valerianol, trans-valeric acid ester, ylangenol, and (-)-isolongifoliol.

[0014] A method for fermenting and enhancing the fragrance of agarwood, comprising the following steps:

[0015] The bacteria Phaeoacremonium scolyti is inoculated into a culture medium, and a fermentation liquid is obtained by culturing, and then the fermentation liquid is sprayed on agarwood powder and fermented to obtain a fermentation product rich in sesquiterpenoids and chromones.

[0016] The agarwood is the resin-containing wood of the plant Aquilaria sinensis of the Thymelaeaceae family; preferably, the agarwood is the agarwood after the agarwood is formed.

[0017] The agarwood powder is powder that is crushed and sieved through a 30-50 mesh sieve.

[0018] The incense is natural or artificial.

[0019] The incense has been 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 culture condition is to culture until OD600 is greater than 2.

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

[0023] The fermentation condition is culturing at 26-30° C. for at least 3 days.

[0024] Compared with the prior art, the present invention has the following advantages and effects:

[0025] The present invention screens out an agarwood symbiotic bacteria from agarwood, and identifies it as a strain of the genus Phaeoacremonium through morphology and molecular biology, and names it Phaeoacremonium scolyti CX-2. The bacteria can ferment the agarwood powder after agarwood formation to produce active substances, and with the increase of fermentation time, the content of agarwood alcohol extract, the content of agarwood characteristic substances, and the antioxidant activity of agarwood alcohol extraction are significantly improved, which proves that the bacteria can produce sesquiterpenes and chromones using wood as raw materials, and has broad application prospects in the industrial production of agarwood. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The morphology of Phaeoacremonium scolyti CX-2 in culture medium; left picture: front side; right picture: back side.

[0027] Figure 2 The spore and hyphae morphology of Phaeoacremonium scolyti CX-2 under a microscope; upper picture: spore morphology; lower picture: hyphae morphology.

[0028] Figure 3 This is the phylogenetic tree of Phaeoacremonium scolyti CX-2.

[0029] Figure 4 These are photos of agarwood raw materials; left: uncrushed agarwood; right: crushed agarwood.

[0030] Figure 5 This is the result of the DPPH antioxidant activity experiment on agarwood alcohol extract. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0032] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0033] Example 1 Isolation, Purification and Identification of Phaeoacremonium scolyti

[0034] Fresh agarwood samples (ensure there is no disease or insect pest) collected from healthy white wood agarwood trees in Dongguan Xiangbo Park were dried in the shade for one year and cut into small cubes with a side length of about 1 cm. After surface disinfection, the sample was cut and the inside was close to the PDA plate. After the plate was sealed, it was placed in a (28±1)℃ constant temperature incubator for culture. After the culture medium was full of hyphae, the strain was purified by hyphae tip inoculation method, and repeated 3 to 4 times until a pure culture strain was obtained. Finally, one of the strains that was easy to culture and had stable transmission characteristics was selected as the target strain, and the strain was numbered CX-2.

[0035] The target strain CX-2 was inverted and cultured on a PDA plate at (28±1)℃ for 7 days, and the colony morphology was observed. Figure 1 ). Take the bacterial suspension and observe the morphology of its spores and hyphae under a microscope ( Figure 2 ). The colony of strain CX-2 is small, reddish brown, nearly round, with white edges. There are diaphragms in the hyphae that separate the hyphae into several segments, each of which contains cytoplasm and one or more nuclei; there are many spores, which are oval in shape. Combined with the "Handbook of Fungal Identification", CX-2 was preliminarily determined to be Sphaeriales. In order to further determine the affiliation of the fungus, the 18S rDNA was sequenced and identified using molecular biological methods. 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] After 18S rDNA nucleotide sequence analysis, the length of the strain's 18S rDNA nucleotide sequence was measured to be 599bp (SEQID No.1). The target strain's 18S rDNA nucleotide sequence was spliced ​​and compared with the 18S rDNA nucleotide sequences of known species in the Gen Bank database on the NCBI website using the Blast program, and a phylogenetic tree was constructed using Mega7 software. The 18S rDNA nucleotide sequence of the strain was compared with the 18S rDNA nucleotide sequences of known species in the Gen Bank database using the Blast program on the NCBI website. The results showed that the strain was most closely related to Phaeoacremonium scolyti, with a gene homology of 99.50%. The evolutionary 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] In view of the above identification results of strain CX-2, CX-2 was determined to be a new strain of Phaeoacremonium scolyti, named Phaeoacremonium scolyti CX-2. The bacteria was deposited in Guangdong Microbiological Culture Collection Center on November 6, 2024, with the deposit number: GDMCCNO.65425. The address of the deposit unit is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0043] Example 2 Agarwood Fermentation and Fragrance Enhancement Experiment

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

[0045] (2) Take resin-containing wood of Aquilaria sinensis of the Thymelaeaceae family (purchased from Dongguan Xiangcheng Industrial Co., Ltd.), age it for one year after harvesting it, grind it and pass it through a 40-mesh sieve; weigh 100 g of agarwood powder, spray the fermentation liquid obtained in step (1) onto the agarwood powder at a solid-liquid ratio of 2:1 (g:ml), and place the inoculated agarwood powder in a constant temperature incubator at (28±1)°C for 3 days.

[0046] Example 3

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

[0048] Example 4

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

[0050] Comparative Example 1

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

[0052] Example 5

[0053] The fermented agarwood was placed in a 45°C oven for 24 hours, and the content of its alcohol-soluble extract was determined according to the cold soaking method in the Pharmacopoeia of the People's Republic of China (2015 edition): about 4g of the fermented and dried agarwood sample was taken, accurately weighed, placed in a 250ml conical flask, accurately added 100ml of 95% ethanol, sealed, cold soaked, shaken from time to time within the first 6 hours, and then allowed to stand for 18 hours, quickly filtered with a drying filter, accurately measured 20ml of the filtrate, placed in an evaporating dish that had been dried to constant weight, evaporated to dryness on a water bath, dried at 105°C for 3 hours, cooled in a desiccator for 30 minutes, and quickly and accurately weighed. The content (%) of the alcohol-soluble extract in the test sample was calculated using the dried product.

[0054] Alcohol extract content:

[0055]

[0056] Note: x——Alcohol extract content (%)

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

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

[0059] M——is the sample mass (g).

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

[0061] Table 3 Agarwood alcohol extract content

[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] The experimental results show that the alcohol extract content in Examples 2 to 4 after fermentation with bacterial liquid is significantly increased compared with that of unfermented agarwood, proving that the bacteria can increase the alcohol extract content of agarwood, and within a certain fermentation time, the effect of increasing the alcohol extract content of agarwood is better as the fermentation time increases. Compared with Comparative Example 1, the fermentation of Phaeoacremoniumscolyti bacterial fermentation liquid can increase the content of agarwood alcohol extract by 1% to 20%; compared with unfermented, the fermentation of Phaeoacremoniumscolyti bacterial fermentation liquid can increase the content of agarwood alcohol extract by 8.02% to 28.34%. Compared with unfermented, the comparative example shows that the addition of sterile water fermentation does not significantly increase the content of agarwood alcohol extract.

[0064] Example 6

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

[0066] GC-MS sample preparation:

[0067] Take 25 ml of anhydrous ethanol to dissolve the alcohol extract, pass it through a 0.45 μm microporous filter membrane, and prepare the sample to be tested by GC-MS.

[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°C to 140°C at a rate of 3°C / min; then linear program temperature increase from 140°C to 160°C at a rate of 1°C / min; then linear program temperature increase from 160°C to 280°C at a rate of 4°C / min, and hold for 10 minutes;

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

[0072] d) Inlet temperature: 250°C;

[0073] e) Detector temperature: 280°C;

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

[0075] g) Injection volume: 1.0 μL;

[0076] h) Split ratio: 10:1;

[0077] i) ion source temperature 230°C;

[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 Analysis of components of agarwood 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, 6,7-dimethoxy-2-(phenylethyl) chromone; sesquiterpenes include β-santalol, azurene, aristolochene, (-)-guaiacol, eucalyptol, costus alcohol, β-humulene, germacron, cedarwooddiol, longifol, β-eudesmol, eucalyptol, dehydrofuranone, carnation Enal, Arimofenone, Isospathulenol, Hydroxyvaleric Acid, Eucalyptol, Ambroxan Oxide, Mosdone, Epoxy Aristolochone, alpha-Cyperone, Curcumol, Inulinolactone, (-)-Thujopsis, Vetiselinenol, 6,9-Guaiadiene, 4(15),5,10(14)-Germagometriene-1-ol, Longgantenone, Trans-Valeric Acid, Ylangenol, (-)-Isolophylline.

[0088] The experimental results show that with the increase of fermentation time, the total content of terpenoids and chromones in agarwood in Examples 2 to 4 generally show an increasing trend. It can be seen from Example 4 in Table 4 that under the action of the fermentation liquid of Phaeoacremoniumscolyti, the content of characteristic substances in agarwood oil can be significantly increased. The total content of sesquiterpenoids in the fermented agarwood oil is 2.86 mg / ml and the total content of chromones is 3.28 mg / ml. Compared with Comparative Example 1, the total content of sesquiterpenoids increased by 27.11% and the total content of chromones increased by 13.49%. Compared with unfermented, the total content of sesquiterpenoids increased by 31.80% and the total content of chromones increased by 13.10%. Compared with unfermented, the total content of sesquiterpenoids and chromones in agarwood alcohol extract did not change significantly in Comparative Example 1.

[0089] Example 7 DPPH Antioxidant Determination of Agarwood Alcohol Extract

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

[0091] The experimental results are as follows Figure 5 As shown, it can be seen that with the increase of fermentation time, the antioxidant capacity of DPPH in the agarwood alcohol extract is stronger. Compared with the unfermented, the antioxidant capacity of the embodiment increased by 1.01% to 2.41%; compared with comparative example 1, the antioxidant capacity of the embodiment increased by 0.31% to 1.38%.

[0092] In summary, the Phaeoacremonium scolyti CX-2 fungus in the present invention can use the wood powder of the Thymelaeaceae plant for fermentation to produce active substances, and with the increase of fermentation time, the content of linalool extract, the content of agarwood characteristic substances, and the antioxidant activity of linalool extraction are significantly improved, which proves that the fungus can produce sesquiterpenes and chromones using wood as raw material, and has broad application prospects in the industrial production of agarwood.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Phaeoacremonium scolyti, characterized in that: The name is Phaeoacremonium scolyti CX-2, which comes from agarwood samples and is obtained through screening and enrichment. The preservation number is GDMCC NO.65425. It was deposited in the Guangdong Provincial Microbial Culture Collection Center on November 6, 2024. The preservation address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The Phaeoacremonium scolyti bacterium according to claim 1, characterized in that: The Phaeoacremonium scolyti bacteria has a small colony on the PDA plate, is reddish brown, nearly circular, and has white edges. The hyphae are divided into several sections by diaphragms, each of which contains cytoplasm and one or more nuclei. The spores are numerous and oval.

3. The Phaeoacremonium scolyti bacterium according to claim 1, characterized in that: The Phaeoacremonium scolyti bacteria can ferment agarwood as a raw material to obtain sesquiterpenes and chromones.

4. Use of the Phaeoacremonium scolyti bacteria according to any one of claims 1 to 3 in the fermentation and aroma enhancement of agarwood.

5. Use of the Phaeoacremonium scolyti according to any one of claims 1 to 3 in the production of sesquiterpenes and chromones.

6. A method for fermenting and enhancing the fragrance of agarwood, characterized in that The steps include: The bacteria Phaeoacremonium scolyti is inoculated into a culture medium, and a fermentation liquid is obtained by culturing, and then the fermentation liquid is sprayed on agarwood powder and fermented to obtain a fermentation product rich in sesquiterpenoids and chromones.

7. The method according to claim 6, characterized in that: The agarwood is the resin-containing wood of the white agarwood plant of the Thymelaeaceae family; The agarwood powder is powder that is crushed and sieved through a 30-50 mesh sieve.

8. The method according to claim 6, characterized in that: The culture medium is PDB liquid culture medium; The culture condition is to culture until OD600 is greater than 2.

9. The method according to claim 6, characterized in that: The ratio of the fermentation liquid to the wood powder is 1-2 mL: 1-2 g.

10. The method according to claim 6, characterized in that: The fermentation condition is culturing at 26-30° C. for at least 3 days.

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