Brevibacterium parafacium and its application in degrading polyphenols in tobacco leaves

Abstract

The present application relates to the field of microorganisms and biotechnology, and particularly relates to a Brevibacillus parafacis and application thereof in degrading polyphenols in tobacco leaves. The Brevibacillus parafacis is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 63066, and the preservation date is December 20, 2022. The strain can effectively degrade the content of polyphenols in cigar tobacco leaves, especially chlorogenic acid.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biotechnology, and in particular to a paraaggregating short bacillus and its application in degrading polyphenols 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] Currently, enzyme preparations and microbial agents are used in cigar fermentation to improve tobacco quality, but the application of polyphenol oxidase and related strains is relatively limited. Studies have shown that adding phenolic substances to wine fermentation can increase aroma compounds such as esters and terpenes. Polyphenols (including chlorogenic acid, scopolamine, and rutin) are important substances affecting cigar tobacco quality; excessive levels can increase bitterness and astringency, affecting the taste. Therefore, chlorogenic acid-degrading strains can be used as microbial agents to improve the aroma and quality of tobacco. Adding them during cigar tobacco fermentation can reduce bitterness, improve and increase aroma, and enhance the industrial usability of tobacco, demonstrating significant market potential and application prospects.

[0004] Currently, most of the bacteria used for treating tobacco leaves belong to the Bacillus genus. For example, patent document CN106579544A discloses the application of a Bacillus subtilis XP strain in degrading polyphenols in tobacco products. Another example is patent document CN106434450A, which discloses a Bacillus subtilis SMXP-58 strain for degrading pectin in tobacco leaves and its application. There are no reports on the use of Brachybacterium paraconglomeratum in tobacco leaves, especially for degrading polyphenols in tobacco leaves. Summary of the Invention

[0005] The present invention aims to solve the above-mentioned problems by providing a paraaggregating short bacillus and its application in degrading polyphenolic substances in tobacco leaves.

[0006] The technical solution to the problem solved by this invention is to first provide a paraconglomeratum, which is deposited at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 63066, deposit date December 20, 2022, and named Brachybacterium paraconglomeratum.

[0007] The inventors of this application screened a strain capable of degrading polyphenols from tobacco leaves and performed colony PCR on the screened strain using universal bacterial primers 27F and 1492R. The PCR products were sent to Songon Biotech in Shanghai for sequencing, and the 16S rRNA sequence was compared using BLAST on NCBI, showing a homology of 99.74%. The nucleotide sequence is shown in SEQ ID NO. 1. The strain was identified as *Brachybacterium paraconglomeratum*. When grown on LB agar, its colony characteristics are: colony diameter 1–2 mm, yellowish-green color, round shape, small and protruding, with a smooth surface.

[0008] In the existing technology, *Bacillus paraaggregatus* is usually used as a microbial feed fermentation agent, with the following main effects: supplementing beneficial bacteria and regulating the animal's microecological balance; producing beneficial metabolites and inhibiting and killing harmful bacteria; having a nutritional effect, promoting animal growth, and improving the body's immune function.

[0009] The inventors of this application unexpectedly discovered that using the above-screened paraaggregate short bacilli in tobacco leaves can effectively degrade polyphenols in tobacco leaves, especially chlorogenic acid in cigar tobacco leaves, thus producing a technical effect that was not anticipated by those skilled in the art.

[0010] Another objective of this invention is to provide the application of *Paracitating Short Bacterium* in the degradation of polyphenols in tobacco leaves.

[0011] Polyphenols include chlorogenic acid, scopolamine, and rutin, etc. The *Paraglossum paraaggregatum* strain of this application exhibits considerable degradation activity against these polyphenols, with particularly good degradation effect on chlorogenic acid. Preferably, the polyphenol is chlorogenic acid.

[0012] The tobacco leaves include cigar tobacco leaves and regular cigarette tobacco leaves. It is worth noting that cigar tobacco leaves and regular cigarette tobacco leaves differ: regular cigarette tobacco leaves are mostly flue-cured, while cigar tobacco leaves are mostly sun-cured; cigar tobacco leaves require two or more fermentation processes, while cigarette tobacco leaves do not. Due to the different processing methods, the composition of cigar tobacco leaves and regular cigarette tobacco leaves differs, and therefore their acceptance of mycorrhizal strains also differs. The *Paraglossum paraaggregates* strain of this application has a particularly good degradation effect on chlorogenic acid in cigar tobacco leaves. Preferably, the tobacco leaves used in this invention are cigar tobacco leaves.

[0013] The para-aggregating short bacillus of this application has an improved effect on degrading polyphenolic substances under fermentation conditions. As a preferred embodiment of the present invention, when applying it, the preparation containing the para-aggregating short bacillus is applied to tobacco leaves and then fermented.

[0014] Among them, the activity concentration of paraaggregating short bacilli in the formulation, the ratio of formulation to tobacco leaves, and fermentation conditions all affect the specific degradation effect.

[0015] The concentration of *Bacillus paraaggregatus* in the formulation should not be too low. Preferably, the concentration of *Bacillus paraaggregatus* in the formulation is at least 1 × 10⁻⁶. 8 CFU / mL. Preferably, the formulation is prepared by the following steps: inoculating the *Paraglomeratus paraaggregatus* into LB liquid medium and culturing at 35℃-40℃ and 200rpm-240rpm for 18h-36h. More preferably, culturing at 220rpm. When preparing the formulation, the inoculum size of *Paraglomeratus paraaggregatus* should be controlled; too much or too little may affect the activity concentration of *Paraglomeratus paraaggregatus*. Preferably, the *Paraglomeratus paraaggregatus* is inoculated into LB liquid medium at an inoculum size of 1.5%-2.5%. More preferably, the inoculum size is 2%.

[0016] 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: 200 g.

[0017] Fermentation conditions affect the efficacy of *Bacillus paraaggregatus*. Preferably, fermentation is carried out at a temperature of 35°C-39°C and a humidity of 68%-72% for 13-17 days. More preferably, fermentation is carried out at a temperature of 37°C and a humidity of 70% for 15 days.

[0018] The beneficial effects of this invention are:

[0019] This invention provides a strain of *Bacillus paraaggregatus* HB001, which, when applied to tobacco leaves, especially cigar tobacco leaves, can effectively regulate the content of polyphenols, especially chlorogenic acid, in cigar tobacco leaves. It can reduce unpleasant tastes such as bitterness and astringency, while also improving the permeability of tobacco leaves, reducing off-flavors, enhancing sweetness, and increasing the quality of tobacco aroma, thereby improving the industrial usability of tobacco leaves. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of *Pseudomonas paraaggregatus* HB001.

[0021] Figure 2 Radar charts showing sensory evaluation of fermented tobacco leaves from Example 2 (C2) and Comparative Example 1 (CK). Detailed Implementation

[0022] 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.

[0023] Experimental materials

[0024] CGA agar medium: Na2HPO4·12H2O 6.15g / L, KH2PO4 1.52g / L, (NH4)2SO4 0.5g / L, MgSO4·7H2O 0.2g / L, CaCl2·2H2O 0.05g / L, trace element solution I 10mL / L.

[0025] Trace element solution I: EDTA 0.5 g / L, FeSO4·7H2O 0.2 g / L, Trace element solution II 100 mL / L.

[0026] Trace element solution II: ZeSO4·7H2O 0.1g / L, MnCl2·4H2O 0.03g / L, H3BO3 0.3g / L, CoCl2·6H2O 0.2g / L, CuCl2·2H2O 0.01g / L, NiCl2·6H2O 0.02g / L, Na2MoO4·2H2O 0.03g / L.

[0027] LB liquid medium: peptone (Oxoid, UK) 10 g / L, yeast extract (Oxoid) 5 g / L, sodium chloride 10 g / L.

[0028] LB solid medium: peptone (Oxoid, UK) 10 g / L, yeast extract (Oxoid) 5 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0029] Example 1

[0030] Isolation, screening, and identification of the paraconglomeratum strain Brachybacterium paraconglomeratum HB001.

[0031] I. Isolation of bacterial strains

[0032] The specific steps are as follows: Take 5g of tobacco leaf sample from Hubei China Tobacco Industry Co., Ltd. and add it to a 500mL shake flask with baffle containing 100mL of 0.9% physiological saline. Elute by shaking at 10℃ and 220rpm for 2.5h. Serially dilute the enrichment culture medium with physiological saline. After dilution, take 100μL of the 10... -4 10 -5 and 10 -6 The diluted solution was spread onto CGA agar medium, with three replicates for each gradient. The spread plates were incubated at 37°C for 21 hours. Single colonies were picked and streaked twice on the same medium for purification before being used for cigar tobacco fermentation.

[0033] II. Screening of bacterial strains

[0034] The specific steps are as follows:

[0035] (1) Take 5g of tobacco leaf sample from Hubei China Tobacco Industry Co., Ltd. and add it to a 500mL shake flask with baffle containing 100mL of 0.9% physiological saline. Shake and elute at 10℃ and 220rpm for 2.5h.

[0036] (2) Spread the eluent onto CGA agar medium and incubate at 37°C for 36 h. Select single colonies of the strain and inoculate them into LB liquid medium and incubate at 37°C and 220 rpm for 21 h.

[0037] III. Identification of Strains

[0038] The specific steps are as follows:

[0039] (1) The 16S rRNA gene of the strain obtained in step 2 was amplified using universal bacterial primers 27F and 1492R. The sequence of universal bacterial primer 27F is 5′-AGAGTTTGATCMTGGCTCAG-3′, and the sequence of universal bacterial primer 1492R is 5′-GGTTACCTTGTTACGACTT-3′.

[0040] (2) PCR was performed using 20 μL of reaction mixture. Taq DNA Polymerase (Takara) was selected for the PCR. 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 and 40 s, to obtain the PCR product.

[0041] (3) The PCR products obtained in step (2) were analyzed by agarose gel electrophoresis and sent to Shanghai Songon Company for sequencing.

[0042] (4) The sequence was compared with previously published bacterial 16S rRNA sequences in the NCBI database using BLAST, and the homology was 99.79%. The nucleotide sequence is shown in SEQ ID NO.1. The colony characteristics of this strain in LB solid medium were: colony diameter 1-2 mm, yellowish-green color, round shape, small and protruding, smooth surface, as shown in... Figure 1 As shown.

[0043] (5) The strain was identified as Brachybacterium paraconglomeratum, belonging to the genus Brachybacterium, and was deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC 63066; it was named Brachybacterium paraconglomeratum HB001.

[0044] Example 2

[0045] Application of Paraaggregating Short Bacterium HB001 strain in cigar tobacco fermentation.

[0046] The specific steps are as follows:

[0047] (1) The strain was inoculated into LB liquid medium at a 2% inoculum and cultured at 37°C and 220 rpm for 24 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 thorough mixing, the preparation was obtained. At this point, the concentration of *Bacillus paraaggregatus* 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 200g of Hubei Laifeng CX81 tobacco leaves in the middle section.

[0048] (2) Place the tobacco leaves sprayed with the fermentation liquid preparation in a self-sealing bag and reflux them at room temperature for 12 hours in a constant temperature and humidity incubator.

[0049] (3) Set the temperature of the constant temperature and humidity incubator to 37℃ and the humidity to 70%, and ferment for 15 days, during which the tobacco leaves are turned over evenly every 7 days.

[0050] (4) After fermentation, store at -20℃.

[0051] Example 3

[0052] Application of Paraaggregating Short Bacterium HB001 strain in cigar tobacco fermentation.

[0053] The specific steps are as follows:

[0054] (1) The strain was inoculated into LB liquid medium at an inoculum rate of 1.5% and cultured at 35℃ 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. After thorough mixing, the preparation was obtained. At this point, the concentration of *Bacillus paraaggregatus* 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 200g of Hubei Laifeng CX81 tobacco leaves in the middle section.

[0055] (2) Place the tobacco leaves sprayed with the fermentation liquid preparation in a self-sealing bag and reflux them at room temperature for 12 hours in a constant temperature and humidity incubator.

[0056] (3) Set the temperature of the constant temperature and humidity incubator to 35℃ and the humidity to 72% for 17 days of fermentation, during which the tobacco leaves are turned over evenly every 7 days.

[0057] (4) After fermentation, store at -20℃.

[0058] Example 4

[0059] Application of Paraaggregating Short Bacterium HB001 strain in cigar tobacco fermentation.

[0060] The specific steps are as follows:

[0061] (1) The strain was inoculated into LB liquid medium at an inoculum rate of 2.5% and cultured at 40℃ and 240rpm for 18h to obtain the fermentation broth. The fermentation broth was added to sterile water at a rate of 4mL to make a final volume of 50mL. After thorough mixing, the preparation was obtained. At this point, the concentration of *Paraglomerulosa* 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 200g of Hubei Laifeng CX81 tobacco leaves in the middle section.

[0062] (2) Place the tobacco leaves sprayed with the fermentation liquid preparation in a self-sealing bag and reflux them at room temperature for 12 hours in a constant temperature and humidity incubator.

[0063] (3) Set the temperature of the constant temperature and humidity incubator to 39℃ and the humidity to 68%, and ferment for 13 days, during which the tobacco leaves are turned over evenly every 7 days.

[0064] (4) After fermentation, store at -20℃.

[0065] Example 5

[0066] Application of Paraaggregating Short Bacterium HB001 strain in cigar tobacco leaves.

[0067] The specific steps are as follows:

[0068] (1) The strain was inoculated into LB liquid medium at a 2% inoculum and cultured at 37°C and 220 rpm for 24 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 thorough mixing, the preparation was obtained. At this point, the concentration of *Bacillus paraaggregatus* 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 200g of Hubei Laifeng CX81 tobacco leaves in the middle section.

[0069] (2) Place the tobacco leaves sprayed with the fermentation liquid preparation in a self-sealing bag, rehydrate them at room temperature for 12 hours in a constant temperature and humidity incubator, and then store them at -20℃.

[0070] Example 6

[0071] This embodiment is basically the same as embodiment 2, except that:

[0072] The fermentation broth was added to sterile water at a rate of 0.4 mL to make a final volume of 46 mL, and the mixture was thoroughly mixed to obtain the formulation.

[0073] Example 7

[0074] This embodiment is basically the same as embodiment 2, except that:

[0075] The preparation was evenly sprayed onto the surface of 500g of Hubei Laifeng CX81 tobacco leaves using a spray bottle.

[0076] Example 8

[0077] This embodiment is basically the same as embodiment 2, except that:

[0078] In step (3), the temperature of the constant temperature and humidity incubator is set at 30℃ and the humidity at 80%, and the fermentation is carried out for 15 days, during which the tobacco leaves are turned over evenly every 7 days.

[0079] Example 9

[0080] Application of Paraaggregating Short Bacillus HB001 strain in flue-cured tobacco leaves.

[0081] The specific steps are as follows:

[0082] (1) The strain was inoculated into LB liquid medium at a 2% inoculum and cultured at 37°C and 220 rpm for 24 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 thorough mixing, the preparation was obtained. At this point, the concentration of *Bacillus paraaggregatus* in the preparation was at least 1 × 10⁻⁶. 8 CFU / mL. Use a spray bottle to evenly spray the preparation onto the surface of 200g of tobacco leaves.

[0083] (2) The tobacco leaves sprayed with the fermentation liquid preparation were first heated to 37°C at a rate of 1°C / h and kept for 24h; then heated to 41°C at a rate of 0.5°C / h and kept for 10h; then heated to 47°C at a rate of 0.4°C / h and kept for 15h; then heated to 53°C at a rate of 0.7°C / h and kept for 14h; then heated to 60°C at a rate of 1°C / h and kept for 3h; and finally heated to 68°C at a rate of 1°C / h and kept for 18h.

[0084] (3) After baking, store at -20℃.

[0085] Comparative Example 1

[0086] The specific steps are as follows:

[0087] (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.

[0088] (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.

[0089] (3) Set the temperature of the constant temperature and humidity incubator to 37℃ and the humidity to 70%, and ferment for 15 days, during which the tobacco leaves are turned over evenly every 7 days.

[0090] (4) After fermentation, store at -20℃.

[0091] Sensory evaluation

[0092] Sensory evaluations were performed on the fermented cigar tobacco leaves in Example 2 and Comparative Example 1.

[0093] The specific steps are as follows:

[0094] (1) Fermented tobacco leaves are rehydrated in a vacuum rehydration machine and then rolled into cigars for sensory evaluation.

[0095] (2) Sensory evaluation 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 1-5, with no zero scores assigned. Qualitative indicators included bitterness, sweetness, saltiness, spiciness, and astringency. Quantitative indicators included smoke concentration, smoke intensity, permeability, smoothness, cigar style prominence, aroma quality, aroma quantity, off-flavors, irritation, aftertaste, sweetness, combustibility, and grayness.

[0096] The results are as follows Figure 2 As shown, it can be seen that the cigar tobacco leaves C2 fermented with the strain fermentation liquid provided by the present invention have better aroma penetration, improved aftertaste and sweetness, increased sweetness, less irritation from off-flavors, reduced bitterness, spiciness and astringency, and improved industrial usability of the tobacco leaves compared with the comparative example 1CK.

[0097] Component analysis

[0098] Untreated blank tobacco leaf samples, as well as treated tobacco leaf samples from the examples and comparative examples, were tested according to "YC-T202-2006 Determination of polyphenolic compounds chlorogenic acid, scopolamine and rutin in tobacco and tobacco products". The test results are shown in Table 1 below.

[0099] Table 1.

[0100] Blank example 10.21 0.33 7.25 Example 2 6.64 0.25 5.73 Example 3 6.85 0.26 5.87 Example 4 6.74 0.26 5.80 Example 5 8.98 0.31 7.03 Example 6 8.68 0.29 6.74 Example 7 7.86 0.29 6.53 Example 8 7.45 0.28 6.31 Example 9 8.47 0.30 6.24 Comparative Example 1 9.90 0.32 7.13

[0101] As shown in Table 1, compared to the blank example, in Examples 2-4 of this application, the degradation rate of chlorogenic acid was around 30%, and the degradation rates of hyoscyamine and rutin were around 20%. In contrast, the degradation rate of polyphenols in Comparative Example 1, which only involved spraying sterile water, was only around 2%. This indicates that the *Bacillus paraaggregatus* and its application scheme of this application can effectively degrade polyphenols in cigar tobacco leaves, with a particularly good degradation rate for chlorogenic acid. In Example 5, the tobacco leaves were not fermented, and although the degradation rate was reduced, it still achieved a certain degradation effect. In Examples 6, 7, and 8, the degradation rate was relatively reduced, indicating that the reduced concentration of *Bacillus paraaggregatus* in the formulation, the reduced amount of the formulation on the tobacco leaves, and the changes in fermentation conditions all affected the degradation effect to some extent. Furthermore, Example 9 shows that *Bacillus paraaggregatus* can also effectively reduce polyphenols in flue-cured tobacco leaf processing.

[0102] 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 paraaggregating short bacillus, characterized in that: The aforementioned *Brachybacterium paraconglomeratum* HB001 is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 63066) on December 20, 2022.

2. The application of *Paraclusobacterium paraaggregatum* as described in claim 1 in the degradation of polyphenols in tobacco leaves; wherein the polyphenols are chlorogenic acid, scopolamine, and rutin.

3. The application of *Pseudomonas paraaggregatus* as described in claim 2 in the degradation of polyphenols in tobacco leaves, characterized in that: The polyphenolic substance is chlorogenic acid.

4. The application of *Pseudomonas paraaggregatus* as described in claim 2 in the degradation of polyphenols in tobacco leaves, characterized in that: The tobacco leaves are cigar tobacco leaves.

5. The application of *Pseudomonas paraaggregatus* as described in claim 2 in the degradation of polyphenols in tobacco leaves, characterized in that: Includes the following steps: The preparation containing the aforementioned paraaggregating short bacilli was applied to tobacco leaves and then fermented.

6. The application of *Pseudomonas paraaggregatus* as described in claim 5 in the degradation of polyphenols in tobacco leaves, characterized in that: The concentration of *Bacillus paraaggregatus* in the formulation is at least 1 × 10⁻⁶. 8 CFU / mL.

7. The application of *Paracitating Short-shaped Bacillus* as described in claim 6 in the degradation of polyphenols in tobacco leaves, characterized in that: The formulation is prepared by the following steps: the paraaggregating short bacillus is inoculated into LB liquid medium and cultured at 35℃-40℃ and 200rpm-240rpm for 18h-36h.

8. The application of *Pseudomonas paraaggregatus* as described in claim 7 in the degradation of polyphenols in tobacco leaves, characterized in that: The paraaggregating short bacilli were inoculated into LB liquid medium at an inoculum of 1.5%-2.5%.

9. The application of *Paraglossobacterium paraaggregatum* as described in claim 6 in the degradation of polyphenols in tobacco leaves, characterized in that: The ratio of the preparation to tobacco leaves is (40-50) mL: 200 g.

10. The application of *Paraglossobacterium paraaggregatum* as described in claim 5 in the degradation of polyphenols in tobacco leaves, characterized in that: Ferment at 35℃-39℃ and 68%-72% humidity for 13-17 days.

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