Bacillus cereus capable of bioconverting limonene

Abstract

The application belongs to the technical field of microbial fermentation, and relates to a Bacillus cereus capable of biotransforming limonene, which is preserved in China Center for Type Culture Collection, is named as Bacillus cereus GJ-02, has a preservation number of CCTCC M 20251438, and a preservation date of June 23, 2025; the application separates and screens the Bacillus cereus strain capable of biotransforming limonene and having high conversion capacity, provides a new strain resource for microbial conversion of D-limonene, and provides a theoretical basis for biosynthesis and metabolic engineering modification of terpenoids, so that the application has a wide application prospect in the field of microbial fermentation.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and relates to a Bacillus cereus that can biotransform limonene. Background Technology

[0002] Limonene, an important functional monoterpene, is widely distributed in various plant essential oils, especially abundant in the essential oils of citrus fruits (particularly their peels). This compound has significant applications in food, fragrances, and pharmaceuticals, and its oxygenated derivatives typically have higher economic value. However, due to its instability, susceptibility to oxidation, and relatively limited aroma profile, its applications remain somewhat restricted. High-value conversion of limonene is currently one of the important pathways for its utilization.

[0003] Carvyl alcohol, also known as carvyl alcohol, is a precursor to carvone and possesses an aroma similar to mint and parsley. It is an important raw material for high-grade food and cosmetic flavorings. Studies have confirmed its anti-inflammatory, anti-tumor, and antioxidant activities. Currently, carvyl alcohol relies on plant extraction or chemical synthesis. The former is limited by low raw material content and seasonal fluctuations, while the latter suffers from poor chiral selectivity and solvent contamination. Limonene and carvyl alcohol have similar skeletons, and theoretically, it is possible to achieve stable production of carvyl alcohol through the bioconversion of limonene. Furthermore, developing efficient and highly selective microbial strains for the synthesis of carvyl alcohol is crucial for the green upgrading of limonene resources.

[0004] Currently, the microbial conversion efficiency of D-limonene to carvacrol is low, limiting large-scale industrial production. Bacillus cereus possesses unique characteristics in microbial metabolism, and in-depth research on it holds the potential to screen strains that efficiently convert D-limonene to carvacrol, thereby promoting the widespread application of carvacrol in various fields and fostering the development of related industries. Summary of the Invention

[0005] The purpose of this invention is to isolate and screen strains with high transformation capabilities from fresh and decaying citrus peels using D-limonene as a carbon source. Strain identification was performed through morphological observation and 16S rRNA gene sequencing. Gas chromatography-mass spectrometry (GC-MS) was used for qualitative analysis of the transformation products, and the product structure was determined by comparison with standards and spectral analysis. The results provide new strain resources for the microbial transformation of D-limonene and offer a theoretical basis for the biosynthesis and metabolic engineering of terpenoids. In this invention, *Bacillus cereus* GJ-02 was isolated from citrus peel and deposited at the China Center for Type Culture Collection.

[0006] To achieve the above objectives, the technical solution of the present invention is: a Bacillus cereus capable of biotransforming limonene, which is deposited at the China Center for Culture Collection (CCTCC) of Wuhan University, Wuhan City, Hubei Province, and is classified as Bacillus cereus GJ-02, with accession number CCTCC M 20251438 and deposit date of June 23, 2025.

[0007] Preferably, the culture characteristics of the Bacillus cereus are as follows: on LB agar plates, the colonies are white, relatively moist, glossy, opaque, granular, with relatively straight edges; under a microscope, the bacteria are rod-shaped and Gram-positive.

[0008] Preferably, the Bacillus cereus can convert D-limonene into carvacrol within 72 hours.

[0009] The present invention also discloses the application of Bacillus cereus in tobacco processing.

[0010] Preferably, the applications include: aroma optimization and improvement, development of harm reduction functions, and optimization of processing technology. Specifically, this includes:

[0011] Targeted synthesis and enhancement of aroma components. Through the biotransformation of limonene by Bacillus cereus, the transformed product possesses more complex aroma characteristics and can be used as a natural tobacco flavoring to enhance the smoothness and harmony of tobacco, making it suitable for cigarette flavoring.

[0012] Harm reduction function development. Limonene itself has antioxidant and anti-inflammatory properties, and its microbial transformation products may further enhance this function, which can be used to reduce free radicals in smoke or neutralize harmful substances (such as polycyclic aromatic hydrocarbons), thereby reducing the health risks of tobacco products.

[0013] Optimize tobacco fermentation and improve quality. Microbial transformation can be combined with tobacco fermentation processes to regulate the composition of volatile substances in tobacco leaves, accelerate the fermentation process and improve aroma purity, or to further shorten aging time and improve raw material utilization.

[0014] The beneficial effects of this invention are:

[0015] This invention isolates and screens Bacillus cereus strains with high conversion efficiency for biotransformation of limonene from fresh and rotten citrus peels, providing new strain resources for the microbial transformation of D-limonene. At the same time, it provides a theoretical basis for the biosynthesis and metabolic engineering of terpenoids. Therefore, this invention has broad application prospects in the field of microbial fermentation. Attached Figure Description

[0016] Figure 1This diagram shows the number and types of Bacillus cereus strains that can bioconvert limonene according to the present invention.

[0017] Figure 2 Radar graphs of olfactory and palatable flavor of Bacillus cereus GJ-02 after biotransformation according to the present invention;

[0018] Figure 3 The above are colony and cell morphology diagrams of Bacillus cereus GJ-02 of the present invention.

[0019] Figure 4 The present invention provides the Bacillus cereus GJ-0216s DNA gene sequence.

[0020] Figure 5 This is a phylogenetic tree diagram of the Bacillus cereus GJ-0216s rDNA gene sequence of the present invention.

[0021] Figure 6 This is the GC-MS chromatogram of the conversion of Bacillus cereus GJ-02 to limonene according to the present invention;

[0022] Figure 7 This is a chemical structure diagram of the D-limonene metabolite produced by the biotransformation of Bacillus cereus GJ-02 according to the present invention. Detailed Implementation

[0023] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] like Figures 1-7As shown, this embodiment uses fresh and rotten citrus peels, branches, leaves, and nearby soil collected from the Citrus Research Institute of Southwest University in Chongqing as raw materials to screen for limonene-resistant strains. A strain using limonene as a carbon source was obtained. Gram staining microscopy and 16S rDNA sequencing confirmed that this strain is *Bacillus cereus*, classified as *Bacillus cereus* GJ-02, and deposited on June 23, 2025, at the China Center for Culture Collections (CCTCC) of Wuhan University, Wuhan, Hubei Province, with accession number CCTCC M 20251438. On LB agar, the colony morphology of this strain is white, relatively moist, glossy, opaque, granular, with relatively straight edges. Under a microscope, the bacterial cells are rod-shaped, and Gram staining is positive. Using LB medium with 0.5% limonene added, the mixture was cultured in shake flasks at 30℃ and 220 rpm for 24 h and 72 h. After extraction of the supernatant with n-hexane, the transformation products were analyzed by GC-MS. The results showed that Bacillus cereus GJ-02 could convert D-limonene to carvacrol in 72 h.

[0025] Example

[0026] 1. Initial screening of strains:

[0027] 1.1 Sample Collection and Processing: Fresh and rotten citrus peels, branches, leaves, and nearby soil were collected from the Citrus Research Institute of Southwest University in Chongqing. The collected samples were sealed in sample bags. 10g of each sample was placed in 150mL of physiological saline and incubated in a constant-temperature shaker at 30℃ and 180r / min for 20min.

[0028] 1.2 Initial Screening of Limonene-Resistant Strains: 0.3 mL of the above culture medium was transferred to 30 mL of YM medium (1% glucose, 0.5% peptone, 0.3% malt extract, 0.3% yeast extract) and 30 μL (0.1% v / v) of D-limonene. After one week of incubation at 30℃ and 150 rpm on a shaker, the culture was serially diluted, and 0.2 mL was evenly spread onto YM solid medium plates. The growth status of the strains was observed during one week of incubation at 30℃. The grown strains were purified by streak plating, and this process was repeated three times. Gram staining was then performed, and the strains were observed under a microscope to confirm their isolation. (See attached image) Figure 1 As shown, a total of 52 bacterial strains were isolated in the initial screening stage, mainly including Bacillus, Pseudomonas, and Bacillus.

[0029] 2. Screening of limonene-resistant strains: The isolated strains were re-inoculated into 30 mL of YM medium and 600 μL (2%, v / v) of D-limonene. The cultures were incubated at 30 °C and 150 r / min for 48 h. 100 μL of the culture was then spread onto YM solid medium and incubated at 30 °C for 48 h. Growth was evaluated afterward. All strains with satisfactory growth (>30 CFU) were considered resistant to 2% (v / v) limonene.

[0030] 3. Sensory analysis of the transformation products of the strains: The screened strains were activated overnight and inoculated with 1% (v / v) in LB medium (1% tryptone, 1% NaCl, and 0.5% yeast extract). After cultivation at 30℃ and 220 rpm to the logarithmic phase, 0.5% (v / v) D-limonene was added for 24 h and 72 h of cultivation, respectively. After the biotransformation experiment, the fermentation broth of each strain was centrifuged at 8000 rpm for 10 min. The supernatant was extracted with n-hexane, and the upper organic phase was volatilized at room temperature for several hours to obtain the sample. The sample was sent to Shaanxi Tobacco Industry Co., Ltd. for sensory evaluation. The results are attached. Figure 2 As shown, the transformation product of strain Bacillus cereus GJ-02 was found to have a resinous, sweet, woody, spicy, fruity and fresh aroma, with the resinous aroma being more prominent.

[0031] 4. Identification of strains:

[0032] 4.1 Preliminary identification of morphological characteristics: The morphological characteristics, color, size, and Gram staining results of the colonies were observed. Bacillus cereus GJ-02 colonies on LB plates were white, moist, glossy, opaque, granular, and had relatively straight edges. Under a microscope, the bacteria were rod-shaped and Gram staining was positive.

[0033] 4.2 Molecular Biological Identification: Using the strain's genome as a template, the 16S sequence of ribosomal DNA was amplified using universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-TACGGCTACCTTGTTACGACT-3). After verifying the PCR product by agarose gel electrophoresis, a PCR fragment of approximately 1500 bp was obtained. The PCR product was recovered using a DNA purification and recovery kit and sequenced (performed by Shenzhen BGI Genomics Co., Ltd.). The sequencing results were submitted to the NCBI website for BLAST search. The results showed that the strain belonged to the genus Bacillus, with the highest homology (99.93%) with Bacillus cereus, and was named Bacillus cereus GJ-02. Multiple sequence matching was performed using MEGA 6.06 software to construct a phylogenetic tree, as shown in the attached figure. Figure 5 As shown.

[0034] 5. Biotransformation of 0.5% limonene (v / v) by Bacillus cereus GJ-02:

[0035] 5.1 Sample preparation: The method is the same as in Example 2.2. After filtering the sample through a 0.22 μm filter membrane, it was added to a sample vial. Analysis was performed using an Agilent 8890-5977B gas chromatograph-mass spectrometer.

[0036] 5.2 Gas Chromatography Conditions: HP-5MS UI column (30m × 250μm × 0.25μm), high-purity helium as carrier gas, constant flow rate of 1.0mL / min, injection volume of 0.3μL, split injection of 10:1, injection port temperature of 250℃. Temperature program: initial column temperature 80℃, hold time 1min, then increase to 170℃ at a rate of 5℃ / min, hold time 3min.

[0037] 5.3 Mass spectrometry conditions: Electron impact ionization (EI) source, transfer line temperature 280℃, ion source temperature 230℃, quadrupole temperature 150℃, electron energy 70eV. Full scan mode (SCAN) was used, with a mass scan range of m / z 30-500 and a scan frequency of 3.2 scan / s.

[0038] 5.4 Qualitative Analysis: GC-MS was used for analysis and identification. The results were retrieved and analyzed using computer-aided spectroscopy (NIST 23.L) and standard reference materials. Table 1 shows the volatile compound analysis of D-limonene during 72h biotransformation of Bacillus cereus GJ-02:

[0039] Table 1

[0040]

[0041] The ion chromatograms of biotransformation of strain Bacillus cereus GJ-02 at 24 h and 72 h were detected by GC-MS and are shown in the attached figures. Figure 6 As shown. Where δ is the relative intensity, t RThe retention times of each compound peak are shown. A comparison with the NIST23.L database and identification of chemical components using a reference quality spectrum revealed that peak 1 is D-limonene (+); peak 2 is (1R,5R)-rel-carveol (cis-Carveol); and peak 3 is L-carveol (-). Based on a search of the NIST23.L database and the mechanism of biotransformation of limonene, the chemical structure of the D-limonene metabolite from the Bacillus cereus GJ-02 strain was deduced, as shown in the attached figure. Figure 7 As shown, Bacillus cereus GJ-02 bioconverts D-limonene to carvacrol. GC-MS analysis of the volatile compounds generated during the D-limonene bioconversion by Bacillus cereus GJ-02 was performed. Table 1 shows that after 72 hours of bioconversion, 15 volatile compounds were identified, mainly including alkenes, alcohols, and ketones. Bacillus cereus GJ-02 bioconverted 16.29% (1R,5R)-rel-carvacrol and 10.09% L-carvacrol.

[0042] In summary, this invention isolates and screens Bacillus cereus strains with high conversion efficiency capable of biotransforming limonene, providing new strain resources for the microbial transformation of D-limonene. It also provides a theoretical basis for the biosynthesis and metabolic engineering of terpenoids, and can be used for aroma optimization, harm reduction development, and processing technology optimization in tobacco processing.

[0043] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A Bacillus cereus capable of biotransforming limonene ( Bacillus cereus GJ-02, characterized in that, The Bacillus cereus described is deposited at the China Center for Culture Collection, accession number CCTCC NO: M 20251438, on June 23, 2025.

2. The application of Bacillus cereus as described in claim 1 in tobacco processing.

3. The application according to claim 2, characterized in that, The applications include: aroma optimization and improvement in tobacco processing.

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