Brucella capable of converting varenishene to generate noottanone, application of Brucella, perfume containing noottanone and preparation method of perfume

Abstract

The invention relates to a Brucella strain capable of converting Varnishene to generate noottanone, an application of the Brucella strain, a perfume containing noottanone and a preparation method of the perfume, and belongs to the technical field of microorganisms. The Brucella is preserved in the China Center for Type Culture Collection on July 11, 2025, and the preservation number of the Brucella is CCTCC M 20251582. According to the present invention, it is found that 0.6 g / L of Fe < 2 + >, 0.8% (v / v) of DMSO and 920 mg / L of vicenthene are added to an LB culture medium, vicenthene can be efficiently converted into noottanone by using Brucella intermedia HNGD-JT1, and the noottanone yield is 245.32 + / -14.37 mg / L.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Brucella strain capable of converting Valencian orangeene into naringin, its applications, a fragrance containing naringin, and its preparation method. Background Technology

[0002] Valencene is an organic compound with a citrus aroma, belonging to the sesquiterpene class. However, its aroma is relatively weak, which limits its use in fragrances and flavorings and fails to meet market demand. Nootkatone has a strong citrus aroma, appearing as a viscous yellow liquid or crystals at room temperature. It is insoluble in water but readily soluble in organic solvents such as ethanol. Nootkatone has a melting point of 37 ℃ and a boiling point of 276.6 ℃. It possesses different stereoisomers, each exhibiting unique aroma characteristics. The perceptibility threshold of nootkatone in water is as low as 1 μg / L. While emitting a strong grapefruit aroma, it also carries woody and sweet orange notes. Nootkatone shows promising applications in the fragrance and flavoring industry, such as in perfume formulation and food flavoring. Using nootkatone as a flavoring agent in tobacco products can enhance the aroma of cigarettes, enrich their flavor, and reduce the irritation during smoking.

[0003] Natural fragrances and flavorings face limitations due to factors such as geographical environment, species, and climate resources. The production of naringin in the market mainly relies on plant extraction and chemical synthesis. Plant extraction suffers from low content and complex separation and purification processes, resulting in higher product prices. While chemical synthesis can produce naringin in large quantities, it requires the use of environmentally unfriendly oxidants and toxic organic solvents, such as chromium trioxide, cobalt acetylacetonate, heavy metal salts, and chloroform, which can easily cause serious environmental damage. Naringin obtained through chemical synthesis is explicitly defined by European food regulations and is not permitted for use or sale within the category of natural fragrances. The commercialization of synthetic naringin faces significant constraints, encountering multiple obstacles from regulatory levels. To address the resource waste and environmental pollution problems caused by plant extraction and chemical synthesis, microbial-mediated biosynthesis of terpenoids demonstrates unique advantages. Biosynthesis offers greater flexibility in raw material selection, and its production process is greener and cleaner, significantly superior to traditional plant extraction and chemical synthesis methods. The invention patent application with publication number CN112342163A discloses a Burkholderia bacterium and its application. This Burkholderia bacterium uses Valencian citrus as a substrate to convert into naringin. The highest yield of naringin is 67.29 mg / L, while the yield of naringin is relatively low. Summary of the Invention

[0004] The first objective of this invention is to provide a Brucella strain capable of converting Valencian citrusene into naringone, thereby solving the technical problem of low naringone yield when existing strains convert Valencian citrusene into naringone.

[0005] A second objective of this invention is to provide an application of Brucella that can convert Valencian orangeene into naringin.

[0006] A third objective of this invention is to provide a flavoring containing naringin.

[0007] The fourth object of the present invention is to provide a method for preparing a fragrance containing naringin.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Brucella abacterium capable of converting Valencian citrusene into naringin was deposited at the China Center for Type Culture Collection on July 11, 2025, with accession number CCTCC M 20251582.

[0009] Application of Brucella bacteria capable of converting Valencian citrus to naringone in the conversion of Valencian citrus as a substrate to naringone.

[0010] Furthermore, Brucella bacteria capable of converting Valencian citrus into naringone were inoculated into LB medium containing Valencian citrus and cultured to obtain naringone; the LB medium contained metal ions and a co-solvent.

[0011] Furthermore, the metal ion is Fe. 2+ The co-solvent is dimethyl sulfoxide.

[0012] Furthermore, in the LB medium, the final concentration of metal ions is 0.6-1.0 g / L; the volume of the co-solvent is 0.6-1.6% of the volume of the LB medium; the final concentration of Valencian citrus is 900-920 mg / L; and the inoculum amount of Brucella that can convert Valencian citrus into cyperiton is 1-3% of the volume of the LB medium.

[0013] Furthermore, the culture conditions in LB medium containing Valencian citrus were: temperature 35-37 ℃, rotation speed 180-220 r / min, and time 36-48 h.

[0014] The fragrance containing naringin is obtained by biotransformation of Valencia citrus by Brucella bacteria, which can convert Valencia citrus into naringin. The fragrance containing naringin also includes linalool, α-terpineol, carvol, D-carvone, α-ionone, styrane, α-ginsenoside, caryophyllene oxide, and α-cyperone.

[0015] A method for preparing a fragrance containing naringin includes the following steps: inoculating the above-mentioned Brucella bacteria capable of converting Valencian citrus into naringin into LB medium containing Valencian citrus and culturing it; wherein the LB medium contains metal ions and a co-solvent.

[0016] Furthermore, the metal ion is Fe. 2+ The co-solvent is dimethyl sulfoxide; in LB medium, Fe 2+ The final concentration of the methyl sulfoxide is 0.6-1 g / L; the volume of dimethyl sulfoxide is 0.6-1.6% of the volume of LB medium; the final concentration of Valencian citrus is 900-920 mg / L; and the inoculum amount of the Brucella that can convert Valencian citrus into cyperiton is 1-3% of the volume of the LB medium.

[0017] Furthermore, the culture conditions in LB medium containing Valencian citrus were: temperature 35-37 ℃, rotation speed 180-220 r / min, and time 36-48 h.

[0018] The beneficial effects of this invention are: This invention is the first to report Brucella intermedia The degradation of Valencian mantlene by HNGD-JT1 can convert it into the natural fragrance naringin. Naringin possesses typical aroma compounds of citrus fruits, has a very low odor threshold, and exhibits a grapefruit-like scent, thus it is widely used in the commercial fragrance and flavor industry. Besides commercial applications, naringin also has significant research value in medical prevention, insect control, and antibacterial applications.

[0019] This invention synthesizes naringin via a microbial method. The conversion process exhibits high specificity and offers advantages such as mild reaction conditions, rapid reaction speed, high yield, and low cost. Valencia citrusene is an important functional sesquiterpene compound widely found in the peels of citrus fruits. This invention provides a highly efficient solution for utilizing citrus byproducts, avoiding waste and economic losses caused by the spoilage of citrus fruits due to overcrowding, improving raw material utilization, and creating greater economic and social benefits.

[0020] This invention discovered that adding 0.6 g / L of Fe to LB medium... 2+ 0.8% (v / v) DMSO, 920 mg / L Valencia orangeene, Brucella intermedia HNGD-JT1 can efficiently convert Valencian citrusene into naringin, with a naringin yield of 245.32 ± 14.37 mg / L. Attached Figure Description

[0021] Figure 1As in Example 1 Brucella intermedia Phylogenetic tree diagram of strain HNGD-JT1; Figure 2 As in Example 1 Brucella intermedia Growth curve of HNGD-JT1 strain; Figure 3 As in Example 1 Brucella intermedia Colony diagram of strain HNGD-JT1; Figure 4 As in Example 1 Brucella intermedia Optical microscope image of HNGD-JT1 strain; Figure 5 As in Example 1 Brucella intermedia Electron micrograph of strain HNGD-JT1; Figure 6 for Brucella intermedia The optimal conditions for the transformation of HNGD-JT1 strain to produce naringin are shown in the figure, where a represents the effect of metal ion type on naringin yield, b represents the effect of metal ion concentration on naringin yield, c represents the effect of co-solvent type on naringin yield, and d represents the effect of co-solvent concentration on naringin yield. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0023] LB liquid culture medium composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0024] LB solid medium composition: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0025] M9 basal medium composition: Na2HPO4 6.78 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, NH4Cl 1.0 g / L, MgSO4·7H2O 0.493 g / L, CaCl2 0.011 g / L, glucose 4 g / L.

[0026] Example 1 Screening and identification of strains that transform Valencian citrusene into naringin (1) Sample preparation and initial screening of strains Soil samples were collected from orange orchards in Wuhan, orange groves in Orange Isle area of ​​Changsha, pomelo groves in Bairuopu rural area, cornfields in Zhengzhou, and peanut fields in Jiaozuo. Oranges and pomelos were purchased from supermarkets in Zhengzhou and collected after being left to rot naturally. 5 g of each soil and fruit sample was incubated in 100 g of 0.85% physiological saline at 37 ℃ and 200 r / min for 4 h to obtain microbial suspensions.

[0027] (2) Screening of strains resistant to 1% Valencian citrusene Take 1 mL of supernatant from the suspension in (1) and add it to 50 mL of LB liquid medium. Add 500 μL (1% v / v) Valencia orange olefin. Before adding Valencia orange olefin, filter the medium using a 0.22 μm organic microporous membrane. Then, incubate the medium at 37 °C and 200 r / min for 48 h to obtain the bacterial suspension.

[0028] Dilute for plating: Take 1 mL of bacterial suspension and serially dilute with sterile water to a concentration of 10. -5 Take 150 μL of the diluted bacterial suspension and spread it onto LB solid medium. After standing for 10 min, invert the medium and incubate overnight at 37 ℃ to obtain colonies.

[0029] Single colony isolation by streak plating: Select single colonies of different shapes, colors, sizes, and morphologies for streak plating, and incubate them overnight in a 37 ℃ incubator. Store the plates in a 4 ℃ refrigerator and repeat streaking until single colonies are isolated.

[0030] Preservation of strains highly tolerant to Valencian citrus: The purified strain was transferred to LB liquid medium and cultured overnight on a shaker at 37 °C and 120 r / min to obtain a bacterial suspension in LB medium. 500 μL of 40% glycerol and 500 μL of the LB medium bacterial suspension were transferred to 2 mL centrifuge tubes and stored at -80 °C.

[0031] The number of strains tolerant to Valencia citrusene selected is shown in Table 1.

[0032] Table 1. Sources and quantities of strains tolerant to Valencian citrusene

[0033] (3) Screening of strains that use Valencian citrusene as the sole carbon source The strains obtained in (2) were selectively cultured using M9 basal medium, and their OD values ​​were measured. 600The value changes. The specific operation is as follows: Centrifuge the strain preserved in (2) to collect the bacterial cells, then add 20 mL of sterilized 0.85% physiological saline, centrifuge at 6000 r / min for 5 min, discard the supernatant, reconstitute and centrifuge again, repeat 3 times to obtain the bacterial suspension. Aspirate the bacterial suspension into 50 mL of M9 basal medium to ensure OD 600 Initial values ​​were above 0.2. 100 μL (0.2% v / v) of Valencian orange glycoside was added, and the mixture was incubated in a shaker at 30 °C and 200 r / min. OD was measured every 12 h. 600 The strains that can survive using Valencian citrusene were preliminarily identified. These strains were isolated from soil in the citrus orchard of Huazhong Agricultural University in Wuhan, Wuhan.

[0034] (4) Microbial transformation experiment of citrus in Valencia The strain obtained in (3) was transferred to 50 mL of LB liquid medium and cultured at 37 °C and 200 r / min. When the OD of the bacterial culture... 600 Once the value reaches 0.6-0.7, add 0.1% (v / v) Valencia orangeene and continue culturing for 48 h.

[0035] (5) Observation under an optical microscope The strains selected in (3) were activated for 8 h to obtain a bacterial suspension. The suspension was streaked onto LB solid medium in a sterile environment and cultured for 12 h. The growth of the strains was observed, and their morphological characteristics were described according to the "Handbook of Systematic Identification of Common Bacteria". A suitable amount of physiological saline was added to a clean glass slide. Colonies on the slide were picked and fixed. Then, primary staining, mordanting, destaining, and counterstaining were performed sequentially using crystal violet, Gram's iodine, 95% ethanol, and safranin. The Gram-stained strains were then observed morphologically using an optical microscope. The physiological and biochemical properties of the strains were tested using micro-biochemical reaction tubes, including the methyl red test, starch hydrolysis, catalase test, and indole test.

[0036] Table 2. Physiological and biochemical identification results of HNGD-JT1

[0037] (6) Scanning electron microscopy observation Take 50 mL of the activated bacterial suspension from step (5) (1×10⁻⁶) 6The bacterial cells were collected in centrifuge tubes (CFU / mL) and centrifuged at 3000 r / min for 5 min to retain the cells. The cells were washed three times with PBS buffer (pH 7.2). 30 mL of 2.5% (v / v) glutaraldehyde solution was added, and the cells were fixed at 4 ℃ for 4 h before centrifugation. The cells were washed three times with PBS buffer (pH 7.2) and twice with ultrapure water, 15 min each time, and then centrifuged to retain the cells. After dehydration and freeze-drying, the cells were transferred to the electron microscopy room for sample preparation. Conductive adhesive was applied to the aluminum sample stage, and the backing paper was removed. A clean toothpick was used to apply a suitable amount of bacterial powder evenly to the surface of the conductive adhesive. Unadhered powder was blown away with a bulb syringe to ensure firm adhesion to the conductive adhesive, and the sample position was recorded. Cross-contamination between samples was avoided during sample preparation, and the bulb syringe was blown towards areas without sample. After gold sputtering, the bacterial samples were observed under a scanning electron microscope.

[0038] (7) Strain identification Using the strain genes screened in (3) as templates, genomic DNA of the strains was extracted using a kit. Primers used were 27F. and The 16S rDNA gene sequence of the screened strain was amplified using colony PCR. The PCR amplification products were sequenced, and the obtained strain sequence data were compared using BLAST for homology search. After PCR amplification, the strain underwent sequencing, yielding a sequence length of 1374 bp. The sequencing results were compared and analyzed on the NCBI website, confirming the strain as Brucella. Brucella intermedia Named Brucella intermedia The nucleic acid sequence of HNGD-JT1 is shown in SEQ ID NO.1.

[0039] SEQ ID NO.1: (8) Determination of transformation products by HS-SPME-GC-MS The strain obtained in (3) was transferred to 50 mL of LB medium and cultured at 37 °C and 200 r / min. When the OD of the bacterial culture... 600 After the pH reached 0.6-0.7, 0.1% (v / v) Valencian orange glycoside was added, and the culture was continued for 24 h, 48 h, and 72 h to obtain the fermentation broth. The fermentation broth was analyzed using HS-SPME-GC-MS to determine the content of naringin, and the results are shown in Table 2. The fermentation broth was centrifuged at 8000 rpm / min for 10 min, and 5 mL of supernatant was transferred to a 20 mL headspace vial. 1.8 g of sodium chloride was added to promote the thorough analysis of volatile components. 100 μL of cyclohexanone solution with a concentration of 947 μg / mL was added as an internal standard. A rotor was added, and the vial was sealed with a PTFE septum. The temperature of the magnetic stirrer was set to 40 ℃ and maintained for 15 min to complete the system equilibration. After the system was equilibrated, the activated 50 / 30 μm DVB / CAR / PDMS extraction head was inserted into the sample system within the septum. At 300 r / min and 40 ℃, headspace adsorption was performed on the fiber head for 40 min. After adsorption, the extraction head was transferred to the GC-MS inlet for 5 min of desorption. The quantitative determination of naringin was performed using the external standard method. Cyclohexanone was used as an internal standard for the quantitative determination of other products. Based on the relationship between the peak area of ​​other aroma components and the peak area of ​​cyclohexanone, the quantitative calculation of each component was realized. The calculation formulas for the content of each component and the conversion rate of naringin are shown in Equations (1) and (2): Equation (1); Equation (2).

[0040] Table 2 Brucella intermedi Products and concentrations of Valencian orangeene converted by HNGD-JT1

[0041] In Table 2, “ND” indicates that the substance was not detected; RI is the retention index of the substance on the HP-5 column; the difference between the same label letters is not significant (P>0.05), while the difference between different label letters is significant (P<0.05).

[0042] As shown in Table 2, in LB medium containing Valencian citronene, strain HNGD-JT1 can convert Valencian citronene into naringin, α-cyperone, caryophyllene oxide, α-ginsenoside, styrane, α-ionone, D-carvone, carvyl alcohol, α-terpineol, and linalool. The yield of α-ginsenoside was 93.32±27.12 mg / L, the yield of naringin was 87.71±15.84 mg / L, and the yield of α-cyperone was 46.45±7.14 mg / L.

[0043] Example 2 Application of strain HNGD-JT1 in converting Valencian citrusene to terpineone The strain HNGD-JT1 from Example 1 was inoculated into LB medium at an inoculation rate of 1% (v / v) and cultured for 12 h. Then, a Valencian citrus-DMSO mixture was added and cultured for another 48 h to obtain the product. The culture temperature was 37 °C and the rotation speed was 200 r / min. The content of naringin in the product was determined according to the method of Example 1, and the naringin content was 245.32 mg / L. The final concentration of Valencian citrus in the LB medium was 920 mg / L, and the amount of DMSO added was 0.8% of the LB medium volume. 2+ The final concentration was 0.6 g / L.

[0044] Examples 3-4 The process by which strain HNGD-JT1 in Examples 3-4 converts Valencian citronene to terpineone is largely the same as in Example 2. The difference between Examples 3-4 and Example 2 is that in the LB medium of Example 3, Fe... 2+ The final concentration was 0.8 g / L. In the LB medium of Example 4, Fe... 2+ The final concentration was 1.0 g / L.

[0045] Examples 5-7 The process by which strain HNGD-JT1 in Examples 5-7 converts Valencian citrusene into terpineone is largely the same as in Example 2. The difference between Examples 5-7 and Example 2 is that in Example 5, the amount of DMSO added to the LB medium is 0.6% of the LB medium volume; in Example 6, the amount of DMSO added to the LB medium is 1.20% of the LB medium volume; and in Example 7, the amount of DMSO added to the LB medium is 1.60% of the LB medium volume.

[0046] Comparative Examples 1-2 The process by which strain HNGD-JT1 of Comparative Examples 1-2 converted Valencian citronene to terpineone was largely the same as in Example 2. The difference between Comparative Examples 1-2 and Example 2 was that in the LB medium of Comparative Example 1, Fe...2+ The final concentration was 0.2 g / L. In the LB medium of Comparative Example 2, Fe... 2+ The final concentration was 0.4 g / L.

[0047] Comparative Example 3 The process by which strain HNGD-JT1 of Comparative Example 3 converted Valencia citronene into cyperone was roughly the same as that of Example 2. The difference between Comparative Example 3 and Example 2 was that the amount of DMSO added to the LB medium in Comparative Example 3 was 0.2% of the volume of the LB medium.

[0048] Experimental Example 1 Effect of metal ion type on naringin yield The concentration of Zn added was clearly 0.3 g / L. 2+ Ca 2+ Mn 2+ Cu 2+ Fe 2+ and Mg 2+ Metal ions were added to LB liquid medium; HNGD-JT1 was inoculated at 1% (v / v) in LB medium containing metal ions and cultured for 12 h, followed by the addition of 920 mg / L Valencia citrusene, and cultured at 37 ℃ and 200 r / min for 48 h to obtain the fermentation broth. The yield of naringin in the fermentation broth was detected, and the results are as follows. Figure 6 As shown, Fe 2+ It can significantly increase the yield of naringin, with the highest yield reaching 92.12 mg / L.

[0049] Experimental Example 2 Effect of cosolvent type on naringin yield LB medium supplemented with 0.6 g / L Fe 2+ HNGD-JT1 was cultured in a medium at an inoculum of 1% (v / v) for 12 h, then Valencia citrus-cosolvent mixed solution was added, and biotransformation was performed at 37 ℃ and 200 r / min for 48 h to obtain fermentation broth. The yield of naringin in the fermentation broth was detected, and the results are as follows: Figure 6 As shown in the figure, the final concentration of Valencian citronene in LB medium was 920 mg / L. With the addition of 0.6% (v / v) of methanol, ethanol, acetone, glycerol, ethyl acetate, and dimethyl sulfoxide (DMSO) as a co-solvent, the experimental results showed that DMSO significantly promoted the biotransformation of Valencian citronene, with the highest yield of citronene reaching 213.45 mg / L.

[0050] fromFigure 6 ab shows that Fe 2+ It can significantly increase the yield of naringin, with the highest yield reaching 92.12 mg / L, when Fe 2+ At a final concentration of 0.6 g / L, the yield of naringin reached a maximum of 198.12 mg / L. From Figure 6 As can be seen from the CD, DMSO has a significant promoting effect on the biotransformation of Valencian orangeene, with the highest yield of naringin reaching 213.45 mg / L. When the amount of DMSO added is 0.8% of the volume of LB medium, the naringin content reaches the maximum value of 245.32 mg / L.

[0051] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the claims.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A Brucella strain capable of converting Valencian citrusene into naringin, characterized in that, Brucella was deposited at the China Center for Type Culture Collection on July 11, 2025, with accession number CCTCC M 20251582.

2. The application of Brucella bacteria as described in claim 1, which can convert Valencian citrus into naringone, in the conversion of Valencian citrus into naringone using Valencian citrus as a substrate.

3. The application of Brucella bacteria capable of converting Valencian citrusene into naringin according to claim 2, characterized in that, Brucella, which can convert Valencian citrus into naringone, was inoculated into LB medium containing Valencian citrus and cultured to obtain naringone; the LB medium contained metal ions and a co-solvent.

4. The application of Brucella bacteria capable of converting Valencian citrusene into naringin according to claim 3, characterized in that, The metal ion is Fe. 2+ The co-solvent is dimethyl sulfoxide.

5. The application of Brucella bacteria capable of converting Valencian citrusene to naringin according to claim 3 or 4, characterized in that, In LB medium, the final concentration of metal ions is 0.6-1.0 g / L; the volume of the co-solvent is 0.6-1.6% of the volume of the LB medium; the final concentration of Valencian citrus is 900-920 mg / L; and the inoculum size of the Brucella that can convert Valencian citrus into cyperiton is 1-3% of the volume of the LB medium.

6. The application of Brucella bacteria capable of converting Valencian citrusene into naringin according to claim 2, characterized in that, The culture conditions in LB medium containing Valencia orangeene were: temperature 35-37 ℃, rotation speed 180-220 r / min, and time 36-48 h.

7. A flavoring containing naringin, characterized in that, Valencia citrus is obtained by biotransformation of Valencia citrus by Brucella, as described in claim 1, which can convert Valencia citrus into terpinene.

8. The method for preparing the fragrance containing naringin as described in claim 7, characterized in that, The method includes the following steps: inoculating the Brucella bacteria of claim 1, which can convert Valencian citrus into cyperone, into LB medium containing Valencian citrus and culturing it; wherein the LB medium contains metal ions and a co-solvent.

9. The method for preparing a fragrance containing naringin according to claim 8, characterized in that, The metal ion is Fe. 2+ The co-solvent is dimethyl sulfoxide; in LB medium, Fe 2+ The final concentration of the methyl sulfoxide is 0.6-1 g / L; the volume of dimethyl sulfoxide is 0.6-1.6% of the volume of LB medium; the final concentration of Valencian citrus is 900-920 mg / L; and the inoculum amount of the Brucella that can convert Valencian citrus into cyperiton is 1-3% of the volume of the LB medium.

10. The method for preparing a fragrance containing naringin according to claim 8, characterized in that, The culture conditions in LB medium containing Valencia orangeene were: temperature 35-37 ℃, rotation speed 180-220 r / min, and time 36-48 h.

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