Rhizobium sp. and its use in degrading beta-carotene in tobacco leaves

Abstract

The present application relates to the field of microorganisms and biotechnology, and particularly relates to a rhizobium huathuanguense and application thereof in degrading beta-carotene in tobacco leaves. The rhizobium huathuanguense is preserved in the Guangdong Microbial Digital Museum Center, and the preservation number is GDMCC No: 63067, and the preservation date is December 20, 2022. The strain is applied to tobacco leaves, especially cigar tobacco leaves, can effectively reduce the content of carotenoids in the fermentation process of cigar tobacco leaves, increase the content of farnesyl acetone, methyl heptenone, geranyl acetone and other substances, can strengthen the bean aroma, woody aroma and nutty aroma of tobacco leaves, and improve the sensory indexes such as aroma quality, aroma amount, offensive odor and irritability of tobacco leaves.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biotechnology, and in particular to a type of Rhizobium praecox and its application in the degradation of β-carotene in tobacco leaves. Background Technology

[0002] Cigars are tobacco products with a unique aroma. They are produced through processes such as sun-drying, agricultural fermentation, pile fermentation, and aging, resulting in complex aromas including floral, roasted, and nutty notes. In recent years, domestic sales and demand for cigars have gradually increased. However, domestically produced cigars lag behind imported cigars in terms of tobacco aroma and quality. Furthermore, limitations in the domestic cigar industry's development and industrial applicability hinder the growth of domestic cigars and their competitiveness in the international market.

[0003] β-Carotene is a component of the pigments in tobacco leaf plasmids. Its degradation products, such as β-ionone, citral, dihydroactinolone, and β-damascone, possess pharmacological potential including anti-inflammatory, antioxidant, and strong free radical scavenging activities, and are widely used in food, cosmetics, and perfumes. Most β-carotene degradation products have fresh floral and fruity aromas, and their low degradation threshold effectively masks off-flavors in tobacco leaves. They significantly influence the enhancement of the aroma, character, and quantity of tobacco aromas. For example, geraniol, farnesylacetone, and dihydroactinolone are significantly positively correlated with caramel sweetness; megastigmatrienone is significantly positively correlated with hay and sweet aromas, enriching the tobacco aroma with a mild, sweet woody and rose-like fragrance; and methylheptenone is significantly positively correlated with a light sweetness, effectively enhancing the aroma character.

[0004] To improve the aroma and quality of tobacco leaves, numerous studies have explored the use of functional strains to enhance the aroma and quality of lower-grade tobacco. For example, Zhang Xiaorui et al. applied amylase-producing strains to tobacco fermentation, promoting the degradation of starch and nicotine and significantly improving the sensory quality of the tobacco. Wu Xinying et al. applied screened protease-producing Bacillus strains to flue-cured tobacco fermentation, promoting the formation of aroma compounds and enhancing aroma and quality. Qin Yanqing et al. added β-carotene degradation products to cigarettes, improving the aroma quality, significantly increasing aroma quantity, reducing off-flavors and irritation, and improving the aftertaste. This indicates that the oxidative degradation of carotenoids has a positive impact on the aroma quality, aroma quantity, and overall quality of tobacco leaves. Carotenoids can be broken down into various aroma compounds at different positions in their carbon chains under the action of microbial peroxidases, carotenoid-degrading oxygenases, and lipoxygenases. However, current research on the screening and application of carotenoid-degrading strains in tobacco leaves is limited.

[0005] Currently, most of the bacteria used for treating tobacco leaves belong to the Bacillus genus. For example, patent document CN111657536A discloses a method for improving tobacco leaf quality, characterized by: screening bacterial strains with carotenoid degradation capabilities; preparing the screened bacterial strains into a biological agent; and applying the biological agent to tobacco leaf aging to obtain tobacco leaves with carotenoid degradation products; wherein the bacterial strain is *Bacillus pleuropsis* isolated from flue-cured tobacco, and the preservation number of *Bacillus pleuropsis* is GDMCC:61029. Patent document CN114376257A discloses a method for improving tobacco leaf quality through synergistic bacterial-enzyme treatment, wherein the method uses *Bacillus pumilus* and carotenoid 9,10' dioxygenase to synergistically treat tobacco leaves.

[0006] Rhizobium, as a common endophytic fungus and rhizosphere microorganism in tobacco leaves, has multiple functions such as promoting growth and fixing nitrogen, solubilizing phosphorus, and degrading pollutants. However, due to its low abundance and difficulty in cultivation, there are still a large number of endophytic microorganisms that need to be studied, and there are no reports of using Rhizobium in tobacco leaves, especially for degrading carotenoids in tobacco leaves. Summary of the Invention

[0007] The present invention aims to solve the above problems by providing a *Rhizobium praecox* strain and its application in degrading β-carotene in tobacco leaves.

[0008] The technical solution to the problem of this invention is to first provide a *Agrobacteriumpusense*, characterized in that: the *Agrobacteriumpusense* is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 63067, deposit date December 20, 2022, and named *Agrobacteriumpusense*.

[0009] The inventors of this application screened a bacterial strain capable of degrading β-carotene from tobacco leaves and performed colony PCR on the screened β-carotene-degrading strain using universal bacterial primers 27F and 1492R. The obtained PCR products were sent to Songon Biotech in Shanghai for sequencing, and then 16S rRNA sequence alignment was performed using BLAST on NCBI, showing a homology of 99.74%. The nucleotide sequence is shown in SEQ ID NO.1, and the strain was identified as *Agrobacterium pusense*. When grown on LB agar, its colony characteristics are: colony diameter 1-2 mm, milky white color, round shape, small and protruding, with a smooth surface.

[0010] In the prior art, *Rhizobium praecox* is commonly used for nitrogen fixation in plants. The inventors of this application unexpectedly discovered that applying the *Rhizobium praecox* obtained through screening to tobacco leaves effectively promotes the degradation of carotenoids and increases the content of substances such as farnesylacetone and methylheptenone in tobacco leaves, thus producing technical effects unexpected by those skilled in the art.

[0011] Another objective of this invention is to provide an application of *Rhizobium praecox* in the degradation of β-carotene in tobacco leaves. The *Rhizobium praecox* is used to degrade β-carotene in tobacco leaves, increasing the content of β-carotene degradation products, including geraniol, hexahydropyronone, auperyl acetone, dihydroactinolone, farnesyl acetone, and methylheptenone, with the increase in the content of farnesyl acetone and methylheptenone being particularly significant.

[0012] The effect of the *Rhizobium praecox* in this application on degradation increment under fermentation conditions is improved. As a preferred embodiment of the present invention, when applying it, the preparation containing the *Rhizobium praecox* is applied to tobacco leaves and then fermented.

[0013] Among them, the active concentration of *Rhizobium praecox* in the formulation, the ratio of the formulation to tobacco leaves, and the fermentation conditions all affect the degradation increment effect.

[0014] The active concentration of *Rhizobium spp.* in the formulation should not be too low. Preferably, the concentration of *Rhizobium spp.* in the formulation is at least 1 × 10⁻⁶. 8 CFU / mL. Preferably, the formulation is prepared by the following steps: inoculating the *Rhizobium praecoxibaris* in LB liquid medium and culturing it at 35℃-40℃ and 200rpm-240rpm for 18h-36h. More preferably, culturing at 220rpm. When preparing the formulation, the inoculum amount of *Rhizobium praecoxibaris* should be controlled; too much or too little may affect the activity concentration of *Rhizobium praecoxibaris*. Preferably, the *Rhizobium praecoxibaris* is inoculated into LB liquid medium at an inoculum amount of 1.5%-2.5%. More preferably, the inoculum amount is 2%.

[0015] The amount of the preparation applied to the tobacco leaves should not be too much or too little. Too much will cause waste, and too little will result in poor degradation effect. As a preferred embodiment of the present invention, the ratio of the preparation to the amount of tobacco leaves is (40-50) mL: 200g.

[0016] Fermentation conditions affect the efficacy of *Rhizobium tumefaciens*. As a preferred embodiment of this invention, fermentation is carried out at a temperature of 35℃-39℃ and a humidity of 68%-72% for 18-24 days. More preferably, fermentation is carried out at a temperature of 37℃ and a humidity of 70% for 21 days.

[0017] The beneficial effects of this invention are: This invention provides a *Rhizobium praecox* strain that, when applied to tobacco leaves, especially cigar tobacco leaves, can effectively reduce the content of carotenoids during the fermentation process of cigar tobacco leaves, increase the content of substances such as farnesylacetone, methylheptenone, and geraniol, enhance the bean, woody, and nutty aromas of tobacco leaves, and improve sensory indicators such as aroma quality, aroma quantity, off-odors, and irritation. This invention provides research ideas for developing novel characteristic aroma cigar products and industrial microbial preparations for cigars. Attached Figure Description

[0018] Figure 1 A colony morphology diagram of *Rhizobium simonii*. Figure 2 Radar charts showing the sensory evaluation of fermented tobacco leaves in Example 2 (C31) and Comparative Example 1 (CK); Figure 3 Radar charts for evaluating quantitative indicators of fermented tobacco leaves in Example 2 (C31) and Comparative Example 1 (CK). Detailed Implementation

[0019] The following are specific embodiments of the present invention, and the technical solution of the present invention will be further described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0020] Experimental materials Enrichment medium: β-carotene 5 g / L, Na2HPO4·12H2O 6.15 g / L, KH2PO4 1.52 g / L, (NH4)2SO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2·2H2O 0.05 g / L, trace element solution I 10 mL / L.

[0021] Trace element solution I: EDTA 0.5 g / L, FeSO4·7H2O 0.2 g / L, Trace element solution II 100 mL / L.

[0022] Trace element solution II: ZeSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H3BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuCl2·2H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, Na2MoO4·2H2O 0.03 g / L.

[0023] Secondary screening medium: β-carotene 5 g / L, peptone (Oxoid, UK) 10 g / L, sodium chloride 10 g / L LB liquid medium: peptone (Oxoid, UK) 10 g / L, yeast extract (Oxoid) 5 g / L, sodium chloride 10 g / L.

[0024] LB solid medium: peptone (Oxoid, UK) 10 g / L, yeast extract (Oxoid) 5 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0025] Example 1 Isolation, screening, and identification of Agrobacterium pusense strain.

[0026] I. Isolation of bacterial strains The specific steps are as follows: Ten g of CRIOLLO 98, CX14 and E-HABANA 2000 tobacco leaf samples provided by Hubei China Tobacco Industry Co., Ltd. were ground with liquid nitrogen to promote the release of endophytic bacteria. The ground powder was added to 90 mL of LB medium containing 20 g / L CX81 tobacco leaves and cultured at 37℃ or 30℃ and 220 rpm for 48 h with shaking.

[0027] II. Screening of bacterial strains The specific steps are as follows: (1) The culture medium obtained in step one was added to an enrichment medium containing 20 g / L tobacco leaves (Hubei Laifeng CX81 tobacco leaves) at a 2% inoculation rate to enrich microorganisms with the function of degrading β-carotene. The enrichment culture conditions were set at 37℃, 220 rpm, and cultured for 24 h. After enrichment culture, the bacterial pellet was collected by centrifugation at 4000 rpm for 10 min.

[0028] (2) Viable cells in the bacterial pellet of (1) were sorted using PI staining solution, resuspended in PBS, centrifuged, and washed multiple times. The final bacterial concentration was adjusted to OD. 600 =0.1~0.2. Incubate with 1 mL of PI staining solution at 4℃ in the dark for 15 min. Viable cells from the enrichment culture will be sorted into 96-well plates containing 200 μL of enrichment medium and cultured at 37℃ and 170 rpm for 72 h. Cells with β-carotene degradation ability (indicated by a lighter color in the fermentation broth or a lower OD value) will be preliminarily selected. 495 The strain (smaller than the blank control) was inoculated into a 96-well plate containing 800 μL of double screening medium and cultured at 37℃ and 170 rpm for 72 h. After centrifugation at 12000 rpm for 2 min, the fermentation supernatant was collected to detect the OD495 of the fermentation broth and observe the color of the fermentation broth. This allowed for the screening of strains that can efficiently degrade carotenoids. The obtained strains were then purified by streak plating.

[0029] III. Identification of Strains The specific steps are as follows: (1) The 16S rRNA gene of the strain obtained in step 2 was amplified using universal bacterial primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).

[0030] (2) PCR was performed using 30 μL of reaction mixture. Taq DNA Polymerase (Takara) was selected as the enzyme. The PCR conditions were: pre-denaturation at 94 °C for 5 min; amplification phase of 35 cycles, performed at 94 °C for 30 s; 55 °C for 30 s; 72 °C for 1 min 40 s, to obtain the PCR product.

[0031] (3) The PCR products obtained in step (2) were analyzed by 1% agarose gel electrophoresis and sent to Shanghai Songon Company for sequencing.

[0032] (4) The sequence was compared with the previously published bacterial 16S rRNA sequence in the NCBI database using BLAST, and the homology was 100%. The nucleotide sequence is shown in SEQ ID NO.1. The colony characteristics of the strain in LB solid medium were: colony diameter 1 mm, milky white color, round shape, small and protruding, smooth surface, as shown in SEQ ID NO.1. Figure 1 As shown.

[0033] (5) The strain was identified as Agrobacterium pusense, belonging to the Rhizobium family, and was deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC 63067. It was named Agrobacterium pusense.

[0034] The strain obtained in this embodiment is denoted as Pusa rhizobium strain HB002.

[0035] Example 2 Application of Rhizobium HB002 strain in cigar tobacco fermentation.

[0036] The specific steps are as follows: (1) The strain was inoculated into LB liquid medium at a 2% inoculum and cultured at 37℃ and 220 rpm for 21 h to obtain the fermentation broth. The fermentation broth was added to sterile water at a rate of 4 mL to make a final volume of 46 mL. After mixing thoroughly, the preparation was obtained. At this time, the concentration of *Rhizobium spp.* in the preparation was at least 1 × 10⁻⁶. 8 CFU / mL. Use a spray bottle to evenly spray the formulation onto the surface of 200 g of Hubei Laifeng CX81 tobacco leaves from the middle section.

[0037] (2) Place the tobacco leaves sprayed with the fermentation liquid preparation in a self-sealing bag and reflux them at room temperature for 12 h in a constant temperature and humidity incubator.

[0038] (3) Set the temperature of the constant temperature and humidity incubator to 37℃ and the humidity to 70%, and ferment for 21 days, during which the tobacco leaves are turned over evenly every 7 days.

[0039] (4) After fermentation, store at -20℃.

[0040] Example 3 Application of Rhizobium HB002 strain in cigar tobacco fermentation.

[0041] The specific steps are as follows: (1) The strain was inoculated into LB liquid medium at an inoculum rate of 1.5% and cultured at 35°C and 200 rpm for 36 h to obtain the fermentation broth. The fermentation broth was added to sterile water at a rate of 4 mL to make a final volume of 40 mL. The mixture was thoroughly mixed to obtain the preparation. At this point, the concentration of *Rhizobium spp.* in the preparation was at least 1 × 10⁻⁶. 8 CFU / mL. Use a spray bottle to evenly spray the formulation onto the surface of 200 g of Hubei Laifeng CX81 tobacco leaves from the middle section.

[0042] (2) Place the tobacco leaves sprayed with the fermentation liquid preparation in a self-sealing bag and reflux them at room temperature for 12 h in a constant temperature and humidity incubator.

[0043] (3) Set the temperature of the constant temperature and humidity incubator to 35℃ and the humidity to 72% for 18 days of fermentation, during which the tobacco leaves are turned over evenly every 7 days.

[0044] (4) After fermentation, store at -20℃.

[0045] Example 4 Application of Rhizobium HB002 strain in cigar tobacco fermentation.

[0046] The specific steps are as follows: (1) The strain was inoculated into LB liquid medium at an inoculum of 2.5% and cultured at 40℃ and 240 rpm for 18 h to obtain the fermentation broth. The fermentation broth was added to sterile water at a rate of 4 mL to make a final volume of 50 mL. The mixture was thoroughly mixed to obtain the preparation. At this point, the concentration of *Rhizobium spp.* in the preparation was at least 1 × 10⁻⁶. 8 CFU / mL. Use a spray bottle to evenly spray the formulation onto the surface of 200 g of Hubei Laifeng CX81 tobacco leaves from the middle section.

[0047] (2) Place the tobacco leaves sprayed with the fermentation liquid preparation in a self-sealing bag and reflux them at room temperature for 12 h in a constant temperature and humidity incubator.

[0048] (3) Set the temperature of the constant temperature and humidity incubator to 39℃ and 68% and ferment for 24 days, during which the tobacco leaves are turned over evenly every 7 days.

[0049] (4) After fermentation, store at -20℃.

[0050] Comparative Example 1 The specific steps are as follows: (1) Spray 46 mL of sterile water evenly onto the surface of 200 g of Hubei Laifeng CX81 tobacco leaves in the middle using a spray bottle, and label it as CK.

[0051] (2) Place the tobacco leaves sprayed with sterile water in a self-sealing bag and reflux them at room temperature for 12 hours in a constant temperature and humidity incubator.

[0052] (3) Set the temperature of the constant temperature and humidity incubator to 37℃ and the humidity to 70%, and ferment for 21 days, during which the tobacco leaves are turned over evenly every 7 days.

[0053] (4) After fermentation, store at -20℃.

[0054] Sensory evaluation The sensory evaluation of the fermented cigar tobacco leaves in Example 2 and Comparative Example 1 was carried out, and the specific steps are as follows: (1) Fermented tobacco leaves are rehydrated in a vacuum rehydration machine and then rolled into cigars for sensory quality analysis.

[0055] (2) Sensory quality analysis of tobacco leaf samples was conducted by Hubei China Tobacco Industry Co., Ltd. Qualitative and quantitative indicators of the samples were scored on a scale of 1 to 5, with no zero scores. Aroma indicators were divided into: woody aroma, bean aroma, floral aroma, honey sweet aroma, caramel sweet aroma, milky aroma, hay aroma, roasted aroma, resinous aroma, nutty aroma, peppery aroma, and coffee aroma. Qualitative indicators were divided into: bitter, sweet, salty, spicy, and astringent. Quantitative indicators were divided into: smoke concentration, smoke intensity, permeability, smoothness, cigar style prominence, aroma quality, aroma quantity, off-flavors, irritation, aftertaste, sweetness, combustibility, and grayness.

[0056] The results are as follows Figure 2 and Figure 3 As shown, it can be seen that, compared with CK in Comparative Example 1, the woody, bean, and nutty aromas of cigar tobacco leaves C31 fermented with the HB002 strain provided by this invention are enhanced, and the milky and peppery aromas are increased. Many quantitative indicators of tobacco leaves, such as aroma quality, aroma quantity, off-flavors, irritation, and smoothness, are significantly improved compared with CK, thus improving the sensory quality of tobacco leaves and their industrial usability.

[0057] Component analysis The components of the fermented cigar tobacco leaves in Example 2 and Comparative Example 1 were analyzed, and the specific steps are as follows: (1) The content of carotenoid degradation products in fermented tobacco leaves was determined by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME / GC-MS). SPME fiber module (50:30 mm divinylbenzene-carboxy-polydimethylsiloxane) was used. After fermentation, the tobacco leaves were dried at 40℃ and ground into powder. 1.5 g of tobacco powder was placed in a headspace vial, and 2-octanol was added as an internal standard at a concentration of 100 μg•μL. -1 The helium flow rate is 1 mL / min. -1 The oven temperature was fixed at 40℃ for 2 minutes, then increased to 250℃ at 15℃ / min and held for 5 minutes. The ion source temperature was 210℃ and the transfer line temperature was 280℃.

[0058] (2) The computer spectral libraries WILEY 8.0 and NIST14 were used for comparison and analysis of compounds. Carotenoid degradation products with a similarity of more than 800 were selected for analysis, and the content was calculated by absolute quantification. The results are shown in Table 1 below.

[0059] Table 1. Table 1 shows that the content of carotenoid degradation products in fermented tobacco leaves increased by 1.33 times, indicating that strain HB002 can significantly promote the degradation of carotenoids in cigar tobacco leaves during fermentation. Compared with the control (CK), HB002 increased the content of farnesylacetone by 2.58 times and the content of methylheptenone to 0.18 μg•g. -1 The increase in the content of carotenoid degradation products plays an important role in enhancing the aroma of tobacco leaves, improving the quality of tobacco aroma, and reducing off-flavors.

[0060] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A type of *Rhizobium praecox*, characterized in that: The aforementioned *Agrobacterium pusense* HB0002 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63067, and the deposit date is December 20, 2022.

2. The application of *Rhizobium praecox* as described in claim 1 in the degradation of β-carotene in tobacco leaves.

3. The application of *Rhizobium praecoxibaris* as described in claim 2 in the degradation of β-carotene in tobacco leaves, comprising the following steps: The preparation containing the aforementioned *Rhizobium tumefaciens* was applied to tobacco leaves and then fermented.

4. The application of *Rhizobium praecoxibaris* as described in claim 3 in the degradation of β-carotene in tobacco leaves, characterized in that: The concentration of *Rhizobium praecoxibaris* in the formulation is at least 1 × 10⁻⁶. 8 CFU / mL.

5. The application of *Rhizobium praecoxibaris* as described in claim 4 in the degradation of β-carotene in tobacco leaves, characterized in that: The formulation is prepared by the following steps: the *Rhizobium praecox* is inoculated into LB liquid medium and cultured at 35℃-40℃ and 200rpm-240rpm for 18h-36h.

6. The application of *Rhizobium praecoxibaris* as described in claim 5 in the degradation of β-carotene in tobacco leaves, characterized in that: The *Prunella vulgaris* rhizobium was inoculated into LB liquid medium at an inoculum concentration of 1.5%-2.5%.

7. The application of *Rhizobium praecoxibaris* as described in claim 4 in the degradation of β-carotene in tobacco leaves, characterized in that: The ratio of the preparation to tobacco leaves is (40-50) mL: 200 g.

8. The application of *Rhizobium praecoxibaris* as described in claim 3 in the degradation of β-carotene in tobacco leaves, characterized in that: Ferment at 35℃-39℃ and 68%-72% humidity for 18-24 days.

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