Curcuma phaeocaulis inner endophyte and application thereof

Abstract

The application discloses a turmeric endophyte and application thereof. The turmeric endophyte Debaryomyces sp. NAVL3-3 is preserved in the China Center for Type Culture Collection on July 8, 2022, and the preservation number is CCTCC M 20221064. The application of the turmeric endophyte Debaryomyces sp. NAVL3-3 in converting curcumin into tetrahydrocurcumin. The application provides an endophyte for bioconversion of tetrahydrocurcumin, and provides a method for preparing tetrahydrocurcumin through fermentation conversion. Through condition optimization, the yield of tetrahydrocurcumin is increased from 6.25 mg / L to 35 mg / L, thereby creating a foundation for later industrial application.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to a turmeric endophytic bacterium and its applications. Background Technology

[0002] Tetrahydrocurcumin is derived from the hydrogenation of curcumin and is the main active metabolite of curcumin in the body. [1] Numerous studies have shown that... [2-6] Tetrahydrocurcumin possesses pharmacological activities such as antioxidant, anti-inflammatory, lipid-regulating, neuroprotective, anti-tumor, and anti-melanin deposition effects. Currently, tetrahydrocurcumin is mainly processed via palladium on carbon. [7,8] Or platinum-iron-nickel hydroxide [9] Catalytic hydrogenation is an option, but the two catalysts used in this method react violently with hydrogen, are prone to explosion, and are also very expensive. Microbial transformation, on the other hand, offers advantages such as high regioselectivity and stereoselectivity, mild reaction conditions, simple operation, low cost, and environmental friendliness, and is gradually gaining attention.

[10] Zhang Weiyu et al. isolated the *Pichia kudriavzevii* ZJPH0802 strain from soil, which can convert curcumin into tetrahydrocurcumin.

[11] Liu Bin et al. also achieved the biosynthesis of tetrahydrocurcumin by overexpressing G6PDH in Escherichia coli. However, the yields of these methods were not high, possibly due to the antibacterial activity of curcumin itself.

[0003] Plant endophytes are widely present in healthy plant tissues. Through interactions with the host plant, they synthesize metabolically active components such as alkaloids, saponins, quinones, flavonoids, and terpenoids. In 1993, the Stierle group first screened an endophytic fungus that produces paclitaxel from Taxus chinensis—Taxomyces andreanae—which has been widely used in biosynthetic pharmaceuticals.

[12] We tested the content of tetrahydrocurcumin in turmeric rhizomes and found that the highest content was found in sprouted tubers. (SHOJI et al.)

[13] A strain of *Diaporthe* sp. was isolated and screened from turmeric rhizomes that can convert curcumin to tetrahydrocurcumin, but the yield was very low. Therefore, we screened a turmeric endophytic bacterium from turmeric rhizomes of different stages, origins, and parts, and optimized the carbon and nitrogen sources of the culture medium through orthogonal methods to develop a stable and efficient method for the biotransformation of tetrahydrocurcumin.

[0004] References

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[0006] [2]PAN M H,CHEN J W,KONG Z L,et al.Attenuation by Tetrahydrocurcuminof Adiposity and Hepatic Steatosis in Mice with High-Fat-Diet-Induced Obesity[J].J Agric Food Chem,2018,66(48):12685-95.

[0007] [3]MURUGAN P,PARI L.Antioxidant effect of tetrahydrocurcumin instreptozotocin-nicotinamide induced diabetic rats[J].Life Sci,2006,79(18):1720-8.

[0008] [4]TANG X,DONG Q,LI J,et al.Anti-Melanogenic Mechanism ofTetrahydrocurcumin and Enhancing Its Topical Delivery Efficacy Using aLecithin-Based Nanoemulsion[J].Pharmaceutics,2021Jul 31;13(8):1185.

[0009] [5]MAITI P,MANNA J,THAMMATHONG J,et al.Tetrahydrocurcumin Has SimilarAnti-Amyloid Properties as Curcumin:In Vitro Comparative Structure-ActivityStudies[J].Antioxidants(Basel),2021Oct 11;10(10):1592.

[0010] [6] LAI CS, HO CT, PAN M H. The Cancer Chemopreventive and Therapeutic Potential of Tetrahydrocurcumin[J]. Biomolecules, 2020May29; 10(6):831.

[0011] [7] Chen Yi, Liu Yu, Chen Qiang. A method for synthesizing tetrahydrocurcumin, CN114031488A[P / OL]. 2022-02-11.

[0012] [8] Guo Wenhua, Xiao Jinxia, ​​Wang Xiaoying, et al. A method for preparing tetrahydrocurcumin, CN104496779B[P / OL]. 2017-01-11.

[0013] [9] Chen Liang, Xin Xiulan, Wei Qing, et al. A novel method for preparing tetrahydrocurcumin, CN111925284A[P / OL]. 2020-11-13.

[0014]

[10] LIN JK, PAN MH, LIN-SHIAU S Y. Recent studies on the biofunctions and biotransformations of curcumin[J]. Biofactors, 2000, 13(1-4):153-8.

[0015]

[11] ZHANG W, HUANG J, WO

[0016]

[12] STIERLE A, STROBEL G, STIERLE D. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew[J].Science,1993,260(5105):214-6.

[0017]

[13] MAEHARA S, IKEDA M, HARAGUCHI H, et al. Microbial conversion of curcumin into colorless hydroderivatives by the endophytic fungus Diaporthesp. associated with Curcuma longa[J]. Chem Pharm Bull (Tokyo), 2011, 59(8): 1042-4. Summary of the Invention

[0018] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a turmeric endophyte, Debaryomycessp.NAVL3-3.

[0019] Another object of the present invention is to provide the application of this strain.

[0020] The objective of this invention can be achieved through the following technical solutions:

[0021] An endophytic bacterium of turmeric, *Debaryomyces* sp. NAVL3-3, was deposited at the China Center for Type Culture Collection on July 8, 2022, with accession number CCTCC M 20221064.

[0022] The bacterial agent prepared from the endophytic bacterium Debaryomyces sp. NAVL3-3 of turmeric.

[0023] The application of the endophytic bacteria Debaryomyces sp. NAVL3-3 or the bacterial agent in the conversion of curcumin to tetrahydrocurcumin.

[0024] A culture medium for preparing tetrahydrocurcumin by converting curcumin using the endophytic bacterium *Debaryomyces* sp. NAVL3-3, wherein the culture medium is based on PBS at pH 7, with the addition of 50-70 g / L maltose, 1-10 g / L corn steep liquor, 0.1-1 g / L zinc sulfate, 0.3-1.8 g / L potassium ions, and 50-100 mg / L curcumin.

[0025] As a preferred embodiment of the present invention, the culture medium is based on PBS and supplemented with: maltose 55 g / L, corn steep liquor 4 g / L, ZnSO4 0.5 g / L, potassium ions 0.6 g / L, and curcumin 62.5 mg / L.

[0026] A method for preparing tetrahydrocurcumin by converting curcumin using the endophytic bacterium *Debaryomyces* sp. NAVL3-3, involves inoculating the wet cells of *Debaryomyces* sp. NAVL3-3 into the culture medium described in this invention for fermentation culture. The inoculation amount is 40-50 g / L, the culture time is 20-40 h, the temperature is 25-30 °C, and the rotation speed is 180-250 rpm.

[0027] As a preferred embodiment of the present invention, the wet endophytic bacteria Debaryomyces sp. NAVL3-3 of turmeric are inoculated into the culture medium for fermentation culture. The inoculation amount is 45 g / L, the culture time is 30 h, the temperature is 28 °C, and the rotation speed is 200 rpm.

[0028] Beneficial effects:

[0029] This invention provides an endophytic bacterium, *Debaryomyces prosopidis*, for the biotransformation of tetrahydrocurcumin, and a method for preparing tetrahydrocurcumin through fermentation. Through single-factor and orthogonal experiments, the nitrogen source, carbon source, metal ions, bacterial count, curcumin content, and fermentation time of the fermentation medium were optimized, increasing the tetrahydrocurcumin yield from 6.25 mg / L to 35 mg / L, laying the foundation for future industrial applications. Attached Figure Description

[0030] Figure 1 Mass spectrometry identification of fermentation broth products

[0031] A. Mass spectrum of tetrahydrocurcumin standard, with the arrow pointing to the mass spectrum peak of tetrahydrocurcumin;

[0032] Mass spectrometry identification results of fermentation products of B. debaryomyces prosopidis.

[0033] Figure 2 Colony image and microscope image of *Debaryomyces prosopidis* strain

[0034] A. Colony image of *Debaryomyces prosopidis* strain B. Microscopic image of *Debaryomyces prosopidis* strain (400x magnification)

[0035] Information on the preservation of biological materials

[0036] Debaryomyces sp. NAVL3-3, deposited at the China Center for Type Culture Collection (CCTCC) on July 8, 2022, at Wuhan University, Wuhan, China, with accession number CCTCC M 20221064. Detailed Implementation

[0037] Example 1 Endophytic Bacterial Screening Method

[0038] The specific screening method for endophytic bacteria in turmeric roots is as follows: Turmeric rhizomes are thoroughly cleaned with running tap water. The roots are then blotted dry with filter paper or air-dried. First, they are soaked in 75% ethanol for 3 minutes, then in sterile water three times for 3 minutes each time. Next, they are soaked in 5% sodium hypochlorite solution for 3 minutes. Finally, they are rinsed 3-4 times with sterile water to thoroughly remove the disinfectant. A sample of the final rinse water is used as a control. The turmeric rhizomes are then cut into thin slices approximately 5cm × 5cm using sterile scissors. These slices are inoculated onto potato dextrose agar (PDA) plates containing penicillin (100U / mL) and streptomycin sulfate (100U / mL). 4-6 slices are placed per plate and incubated at 28℃. Once bacteria have grown around the plant tissue on the medium, they are transferred to new agar plates and purified using the streak plating method. The purified endophytic bacteria were inoculated into seed culture medium and cultured at 28℃ and 200rpm on a shaker for 48h to obtain the endophytic bacteria NAVL3-3 seed culture.

[0039] Example 2: Identification of Single Colony Strains

[0040] The bacterial strain preserved in Example 1 at -80℃ was activated by adding 1% (by volume) of the strain to 100mL of PDB liquid medium. The medium was then placed in a shaker at 28℃ and 200rpm for 48 hours. After culturing, the bacterial solution was transferred to a centrifuge tube and centrifuged in a refrigerated centrifuge to collect the strain. Total genomic DNA was extracted from the strain using a genomic extraction kit. Using universal primers for bacterial 18rRNA, and with the extracted genome as a template, the PCR product was obtained by sequentially denaturing at 95℃ for 1 min, annealing at 50-60℃ for 30 s, and extending at 72℃ for 2-5 min, repeated 35 times. The PCR product was then subjected to gene sequencing to obtain the detection sequence. The obtained PCR product gene sequence was then imported into the NCBI database for Blast sequence comparison to search for homologous species. The most similar species were identified, and the biological classification of the strain was determined to be *Debaryomyces prosopidis*, with a similarity of 98.45%. The strain NAVL3-3 was deposited with CCTCC, with accession number CCTCC M20221064.

[0041] Upstream primer: 5'-TCCGTAGGTGAACCTGCGC-3'(ITS1).

[0042] Downstream primer: 5'-TCCTCCCGCTTATTGATATGC-3'(ITS4).

[0043] Based on the identification of the above strains, the nucleotide sequence of this turmeric endophyte is shown in SEQ ID NO:1.

[0044] Example 3: Method for Expanding the Culture of the Strain

[0045] The turmeric endophytic bacteria strain preserved in a -80℃ refrigerator as described in Example 1 was activated by inoculating 1% (v / v) of the strain into 100mL of PDB liquid medium and cultured on a shaker at 28℃ and 180rpm for 48h. Then, the strain was activated again by inoculating 1% (v / v) of the strain into 1500mL of PDB liquid medium and cultured on a shaker at 28℃ and 180rpm for 48h. The fermentation culture was then placed in a centrifuge tube and centrifuged using a refrigerated centrifuge at 4℃ and 6000rpm for 2min. The supernatant was removed, and the bacterial strain was collected. Then, 30mL of sterile PBS was added, and the tube was centrifuged again in a refrigerated centrifuge to remove the supernatant and wash away any remaining fermentation culture, yielding wet turmeric endophytic bacteria NAVL3-3.

[0046] Example 4: Test for the conversion of curcumin into tetrahydrocurcumin by endophytic bacteria

[0047] 1.5 g of wet NAVL3-3 cells were suspended in 40 mL of phosphate buffer, and 2.5 mg of curcumin substrate was added for transformation. The transformation was carried out at 28 °C, 200 rpm, and 36 h in a shaker. After the reaction, the cells were separated by centrifugation, and the supernatant was extracted three times with ethyl acetate. The extracts were combined, and the ethyl acetate was removed by vacuum distillation. The residue was dissolved in methanol for chromatographic analysis. After transformation, tetrahydrocurcumin was identified by liquid chromatography-electrospray ionization triple quadrupole mass spectrometry, and the content of tetrahydrocurcumin was determined by HPLC ultra-high performance liquid chromatography.

[0048] The conditions for ultra-high performance liquid chromatography (UHPLC) determination are: using C 18 The BEH column was used with an elution system of acetonitrile:0.5% acetic acid = 11:9, a column temperature of 30℃, a flow rate of 0.2 mL / min, and an injection volume of 2 μL.

[0049] Preparation of tetrahydrocurcumin reference standard: Accurately weigh tetrahydrocurcumin reference standard, add an appropriate amount of methanol to dissolve it, and prepare reference standard solutions with concentrations of 250 mg / mL, 125 mg / mL, 62.5 mg / mL, and 31.25 mg / mL.

[0050] The obtained reference standard and test sample were subjected to ultra-high performance liquid chromatography (UHPLC) analysis, and the content of tetrahydrocurcumin after conversion was found to be 30 mg / L.

[0051] Samples were separated by HPLC-ESI triple quadrupole mass spectrometry using a C18 column (50 mm × 2.1 mm, 3 μm particle size). The column temperature was 25 °C, and elution was performed using acetonitrile and 0.1% acetic acid solution (80:20, v / v) at a flow rate of 0.3 mL / min. Each sample run was 3.0 min, and the injection volume was 10 μL. The mass spectrometry parameters were as follows: spray voltage, 4500 V; evaporator temperature, 400 °C; jacket gas pressure, 35 psi; auxiliary gas pressure, 30 psi; capillary temperature, 350 °C; collision gas pressure, 1.5 mTorr; collision energy of tetrahydrocurcumin, 32 eV. The scan time for each analyte was set to 0.3 s. Data acquisition was performed using a Thermo MRM of 1 Channel ES+THC 1.27e5. The mass spectrometry peak chromatogram of the tetrahydrocurcumin standard and the mass spectrometry identification results of the fermentation product of Debaryomyces prosopidis are shown below. Figure 1 .

[0052] Example 5: Culture medium optimization and orthogonal experiment

[0053] Step 1: Screening for optimal carbon source, nitrogen source, and metal ions

[0054] Fermentation media were prepared using PBS at pH 7 with eight carbon sources (50 g / L: glucose, sucrose, lactic acid, citric acid, sodium acetate, glycerol, lactose, and malt extract) and 62.5 mg / L curcumin. The inoculum was 40 g / L, and the culture time was 36 h at 28 °C and 200 rpm. Fermentation media were also prepared using PBS at pH 7 with six nitrogen sources (4 g / L: urea, ammonium sulfate, tryptone, yeast extract, beef extract, and corn steep liquor) and 62.5 mg / L curcumin. The inoculum was 40 g / L, and the culture time was 36 h at 28 °C and 200 rpm. Each treatment was repeated three times. Based on the tetrahydrocurcumin produced during fermentation, the results showed that malt extract was the optimal carbon source and corn steep liquor was the best nitrogen source.

[0055] Step 2: Screening of metal ions

[0056] Following the carbon source screening results in step one, a maltose 20 g / L, PBS (pH 7.0) basal fermentation medium was used, with 62.5 mg / L curcumin added. The inoculum was 40 g / L, and fermentation was carried out for 36 h at 28°C and 200 rpm. Eight inorganic ions (Al(SO4)3, ZnSO4, CaCl2, MnSO4, CuCl2, CoCl2, FeSO4, MgSO4, and KCl) at 0.5 g / L were added to the fermentation medium, with each treatment repeated three times. The fermentation results showed that potassium and zinc ions were the optimal metal ions.

[0057] Step 3: Screening for optimal concentrations of carbon source, nitrogen source, and metal ions

[0058] Fermentation was conducted using PBS (pH 7.0) as the basal medium, with an inoculum of 40 g / L, a culture time of 36 h, a temperature of 28 °C, and a rotation speed of 200 rpm. Malt extract, corn steep liquor, zinc ions, and potassium ions were used as research subjects, and an orthogonal design was performed according to table L9(3). 4) The concentrations of malt extract, corn steep liquor, KCl, and ZnSO4 in the culture medium were optimized. The factor levels are shown in Table 1, and the experimental design is shown in Table 2. Each group had three replicates.

[0059] The range analysis in Table 2 shows that the malt extract concentration has the largest range and is the main factor affecting tetrahydrocurcumin. Corn steep liquor and potassium ions are the main factors, and the order is: malt extract > corn steep liquor > zinc ions > potassium ions. The optimal ratio of the four factors is A2B1C2D3.

[0060] Table 1. Orthogonal Factor Level Table

[0061]

[0062] Table 2 Results of orthogonal experiment L9(34)

[0063]

[0064]

[0065] Step 5: Screening for the optimal bacterial load

[0066] A certain amount of wet bacteria was inoculated into a fermentation medium containing 55 g / L malt extract, 4 g / L corn steep liquor, 0.5 g / L zinc ions, and 0.6 g / L potassium ions, resulting in final wet bacterial concentrations of 20 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 60 g / L, and 70 g / L. Transformation was carried out in PBS (pH 7.0) at 28°C and 200 rpm, with each treatment repeated three times. The optimal bacterial concentration was determined to be 45 g / L based on the amount of tetrahydrocurcumin produced during fermentation.

[0067] Step Six: Screening for the Optimal Amount of Curcumin

[0068] Using 55 g / L malt extract powder, 4 g / L corn steep liquor, 0.5 g / L zinc ions, and 0.6 g / L potassium ions as the fermentation medium in PBS (pH 7), 45 g / L of wet bacterial cells were added. A certain amount of curcumin was then added to the medium at concentrations of 50 mg / L, 62.5 mg / L, 87.5 mg / L, 112.5 mg / L, 137.5 mg / L, 250 mg / L, 500 mg / L, and 1000 mg / L. The mixture was incubated in PBS (pH 7.0) at 28°C for 36 h at 200 rpm. The optimal amount of curcumin was screened, and each treatment was repeated three times. Based on the amount of tetrahydrocurcumin produced during fermentation, the optimal amount of curcumin was found to be 62.5 mg / L.

[0069] Step 7: Screening for the optimal fermentation time

[0070] Using 55 g / L malt extract powder, 4 g / L corn steep liquor, 0.5 g / L zinc ions, and 0.6 g / L potassium ions in PBS (pH 7.0) fermentation medium, with 62.5 mg / L curcumin added, the mixture was incubated at 28℃ and 200 rpm for 24 h, 30 h, 36 h, 48 h, 60 h, and 72 h to screen for the optimal fermentation time. Based on the amount of tetrahydrocurcumin converted during fermentation, the optimal fermentation time was selected as 30 h, achieving a final conversion rate of 53%.

Claims

1. An endophytic fungus of turmeric Debaryomyces sp. NAVL3-3 was deposited at the China Center for Type Culture Collection on July 8, 2022, with accession number CCTCC NO: M 20221064.

2. The turmeric endophytic bacteria as described in claim 1 Debaryomyces sp . Bacterial agent prepared from NAVL3-3.

3. A method utilizing the turmeric endophytic bacteria described in claim 1 Debaryomyces sp The culture medium for converting curcumin to tetrahydrocurcumin using NAVL3-3 is characterized by... The culture medium is based on PBS at pH 7, with the addition of 50-70 g / L maltose, 1-10 g / L corn steep liquor, 0.1-1 g / L zinc sulfate, 0.3-1.8 g / L potassium ions, and 50-100 mg / L curcumin.

4. The culture medium according to claim 3, characterized in that... The culture medium was based on PBS and supplemented with: maltose 55 g / L, corn steep liquor 4 g / L, ZnSO4 0.5 g / L, potassium ions 0.6 g / L, and curcumin 62.5 mg / L.

5. A method utilizing the turmeric endophytic bacteria described in claim 1 Debaryomyces sp The method for preparing tetrahydrocurcumin by converting NAVL3-3 into curcumin is characterized by... The turmeric endophytes as described in claim 1 Debaryomyces sp NAVL3-3 wet cells were inoculated into the culture medium described in claim 3 or 4 for fermentation culture, with an inoculation amount of 40-50 g / L, a culture time of 20-40 h, a temperature of 25-30℃, and a rotation speed of 180-250 rpm.

6. The method according to claim 5, characterized in that... The turmeric endophytes as described in claim 1 Debaryomyces sp NAVL3-3 wet cells were inoculated into the culture medium described in claim 3 or 4 for fermentation culture, with an inoculum amount of 45 g / L, a culture time of 30 h, a temperature of 28℃, and a rotation speed of 200 rpm.

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