A dendritic spore-forming bacillus and its applications

Abstract

This application discloses a dendritic Bacillus strain and its applications, belonging to the field of microbial technology. The strain, classified as *Paenibacillus dendritiformis* yhs-1, is deposited at the China Center for Type Culture Collection (CCTCC) on October 25, 2023, with accession number CCTCC NO: M 20232033. This strain was screened from tobacco leaves and fruit and vegetable samples and exhibits good degradation ability for lutein. When applied to tobacco fermentation, it degrades lutein in tobacco leaves and produces 18 aroma compounds, resulting in a rich aroma and aftertaste without any off-flavors or irritating tastes. The fermented tobacco products, when used in cigarettes, significantly improve the smoking quality of the cigarettes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a tree-like Paenibacillus and application thereof. BACKGROUND

[0002] Lutein, also known as plant lutein, is an oxygen-containing carotenoid, which is widely present in fruits, vegetables, fresh flowers and tobacco plants, and is an important flavor precursor. The content of lutein in grapes can reach about 1 μg / g, and the content of lutein in tobacco leaves is even higher, which can reach about 150 μg / g. Lutein can produce a variety of aroma substances such as β-ionone, β-dihydrodamascenone and maconin under certain conditions. These aroma substances have relatively low threshold values, small irritability and good aroma quality, are the source of plant floral and fruity aroma, and are also important components of wine and tobacco flavor. However, the degradation mode of lutein will affect the types and product quality of the degradation products. For example, after 0.02% lutein is added to tobacco, the aroma, offensive odor and irritability of the tobacco are increased, but the aftertaste is poor. The significant increase in offensive odor and irritability is related to the large amount of benzene and its homologues produced by thermal decomposition of lutein. Therefore, using appropriate technology to degrade lutein in the processing of tobacco will help to reduce the harm of cigarettes and improve the smoking quality.

[0003] At present, the research on lutein degradation is mainly concentrated in the maturation and curing process of tobacco leaves, and there are few reports in other fields. The degradation modes involved are mainly physical degradation (thermal cracking), chemical degradation and oxidative degradation, and the research on biological degradation is few. The microorganisms reported to be able to degrade lutein mainly include Geotrichum sp (white geotrichum) and Enterobacter hormaechei (Hormaechei enterobacter), and the degradation products are β-ionone and 8-methyl-α-ionone, respectively. In addition, A. Sanchez-Contreras et al. screened two strains of Geosmithmin and Bacillus from the flowers of marigold, which can also degrade lutein after mixed fermentation, and the main products are β-ionone, 7,8-dihydro-β-ionone, 3-hydrogen-β-ionone and 7,8-dihydro-β-ionol. From the above reports, it can be found that the biological method for degrading lutein has obvious advantages, the product components obtained are relatively simple, and are recognized as "natural ingredients". Therefore, the biological method for degrading lutein has a wide application prospect. SUMMARY

[0004] To address the aforementioned issues, this application provides a dendritic Bacillus strain. The applicant screened a strain yhs-1 with good lutein degradation ability from tobacco and fruit and vegetable samples, and determined its degradation products. The composition of the culture medium for lutein degradation by strain yhs-1 was optimized through single-factor and response surface methodology experiments. Finally, the strain was applied to tobacco fermentation, laying the foundation for the development of a new biological flavoring process.

[0005] The specific technical solution of this application is as follows:

[0006] This application provides a dendritic spore-forming bacterium, which is classified as Paenibacillus dendritiformis yhs-1 and has been deposited at the China Center for Type Culture Collection (CCTCC) on October 25, 2023, with accession number CCTCC NO: M 20232033.

[0007] This application also provides a bacterial suspension comprising the aforementioned Bacillus dendriticis.

[0008] Optionally, the bacterial suspension is prepared by the following method:

[0009] Pick a single colony and place it in LB liquid medium, then shake and incubate until the bacterial culture reaches OD500. 600 Seed culture was obtained at a concentration of 0.8-1.2. The culture was expanded to obtain bacterial suspension at an inoculation ratio of 1.8-2.2%. The bacterial suspension was centrifuged, the supernatant was discarded and the bacterial precipitate was obtained. The bacterial precipitate was resuspended and washed with sterile PBS to obtain bacterial suspension.

[0010] This application also provides the following applications

[0011] The application of the above-mentioned dendritic Bacillus and / or bacterial suspensions in the degradation of lutein.

[0012] The application of the above-mentioned Bacillus dendriticis and / or bacterial suspensions in tobacco flavoring.

[0013] The application of the above-mentioned Bacillus dendriticis and / or bacterial suspension in tobacco fermentation involves spraying the bacterial suspension of the aforementioned Bacillus dendriticis onto the surface of tobacco shreds for fermentation.

[0014] Optionally, the fermentation conditions are 32-40℃ and 45-65% humidity for 45-60 hours.

[0015] Optionally, the volume-to-mass ratio of the bacterial suspension to the tobacco shreds is 1:45-55.

[0016] Optionally, the concentration of the bacterial suspension is 5 × 10⁻⁶. 7 ~5×10 10CFU / mL.

[0017] This application also provides the application of the above-mentioned Bacillus denticulatus and / or bacterial suspension in improving the quality of tobacco leaves.

[0018] The beneficial effects of this application include, but are not limited to:

[0019] 1. The Bacillus dendriticis strain yhs-1 screened from fruits, vegetables and tobacco leaves in this application is capable of fermenting and degrading lutein in tobacco leaves and producing octadecenal (C 18 H 34 O) and phytol (C 20 H 40 The product contains 18 flavor compounds, including O, which are the result of the degradation and transformation of lutein by strain yhs-1. Because fruits, vegetables and tobacco contain a large number of other nutrients besides lutein, these substances can meet the nutritional needs of strain yhs-1. Under these circumstances, the degradation and transformation of lutein has a more complex metabolic pathway.

[0020] These aromatic substances give the tobacco leaves a rich aroma and aftertaste, and the product is free of other off-flavors and irritating tastes. This enhances the aroma of the tobacco leaves while preventing the production of harmful gases. Fermented tobacco products can significantly improve the smoking quality of cigarettes when used in cigarettes.

[0021] 2. Furthermore, the strain YHS-1 of this application can achieve a degradation rate of 23.66% for lutein in tobacco leaves (from 210.5 μg / g before fermentation to 160.7 μg / g); the fermented tobacco leaves have higher sensory scores, less off-flavors, less pungent taste, a purer and more comfortable aftertaste, and increased aroma. Attached Figure Description

[0022] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0023] Figure 1 This is the lutein standard curve for this application;

[0024] Figure 2 The degradation rate of lutein by the strain of this application;

[0025] Figure 3 The effect of fermentation time on the degradation of lutein by strain YHS-1;

[0026] Figure 4 The colony morphology of strain yhs-1;

[0027] Figure 5 Phylogenetic tree of strain yhs-1;

[0028] Figure 6 The effect of glucose addition on lutein degradation rate;

[0029] Figure 7 The effect of peptone addition on lutein degradation rate;

[0030] Figure 8 The effect of MgSO4 addition on lutein degradation rate;

[0031] Figure 9 The response surface and contour lines show the effect of the interaction between various factors on the degradation rate of lutein.

[0032] Figure 10 To validate the results of culture medium optimization. Detailed Implementation

[0033] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. The present application is further described below with reference to specific embodiments, but the scope of protection of the present application is not limited thereto; unless otherwise specified, all kinds of reagents, instruments, etc. used in the embodiments are commercially available products.

[0034] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the following examples and experimental cases:

[0035] Materials and methods

[0036] Materials and Culture Media

[0037] Materials: Tobacco leaf samples (Inner Mongolia Kunming Tobacco Co., Ltd.), fruit and vegetable samples (Inner Mongolia Agricultural University Supermarket), lutein (Beijing Wanjia Shouhua Biotechnology Co., Ltd.).

[0038] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH=7.0.

[0039] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, pH=7.0.

[0040] Basic fermentation medium: glucose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, MgSO4 0.2 g / L, pH=7.0, sterilized at 121 ℃ for 20 min, then add 1 ml / L lutein solution.

[0041] Lutein solution: 0.2 g lutein, 0.2 g Tween 80, 400 ml CH2Cl2. The solvent was evaporated under reduced pressure and dissolved in 10 ml anhydrous ethanol. The solution was filtered through a 0.22 μm sterile filter.

[0042] Instruments and equipment

[0043] High-performance liquid chromatography (HPLC), Agilent Technologies, Inc.; Gas chromatography-mass spectrometry (GC-MS), Agilent Technologies, Inc.; Clean bench, Shanghai Yiheng Scientific Instruments Co., Ltd.; Autoclave, Shanghai Xinpu Instrument Equipment Co., Ltd.; High-speed centrifuge, Shanghai Chengguan Instrument Co., Ltd.; Shaking incubator, Shanghai Yiheng Scientific Instruments Co., Ltd.

[0044] The specific experimental procedure is as follows:

[0045] 1. Screening of bacterial strains

[0046] The tobacco leaves and fruits and vegetables were ground, and 4 g was weighed and placed into a shake flask containing 50 mL of sterile water. After thorough shaking, the mixture was filtered through gauze. The filtrate was diluted to a suitable gradient and spread onto LB solid agar plates. After incubation at 37 ℃ for 48 h, single colonies were picked and streaked for purification. The purified single colonies were inoculated into LB liquid agar and incubated at 37 ℃ and 180 r / min for 24 h with shaking. 1 mL of the bacterial solution was then stored in glycerol (the applicant screened 198 culturable bacteria from tobacco leaf and fruit and vegetable samples, so all culturable microorganisms were purified and stored first). At the same time, 2% (V / V) of the inoculum was inoculated into the basal fermentation medium and incubated at 37 ℃ and 180 r / min for 48 h to obtain the fermentation broth. The lutein content was measured using high performance liquid chromatography (HPLC), and the lutein degradation rate was calculated according to the formula. Strains with high lutein degradation rates were screened. Lutein degradation rate = [1-(C 实验 / C 对照 )]×100%, where: C 实验 C represents the concentration of lutein in the experimental group. 对照 The concentration of lutein in the control group is expressed in mg / L.

[0047] The method for determining lutein content is as follows:

[0048] The fermentation broth was centrifuged (4℃, 6500 r / min, 10 min) to obtain the supernatant. An equal volume of dichloromethane was added for repeated extraction three times to obtain the lutein extract. The extract was dried overnight with anhydrous Na2SO4, and the solvent was dried using a nitrogen blower. Then, 2 mL of methanol was added, and the mixture was filtered through a 0.22 μm organic filter for high performance liquid chromatography (HPLC) analysis. An equal volume of sterile basal fermentation medium was used as a control.

[0049] The chromatographic column used was a C18 column (4.6 mm × 150 mm, particle size 4 µm), the column temperature was 30℃, the mobile phase was methanol:water (97.5:2.5 v / v), the flow rate was 1 mL / min, the wavelength was 445 nm, and the injection volume was 20 µL.

[0050] Plotting the lutein standard curve:

[0051] Accurately weigh 10 mg of lutein standard into a 10 mL amber volumetric flask, dissolve in anhydrous ethanol, and dilute to the mark to obtain a standard stock solution with a concentration of 1 mg / mL. Transfer 0.125, 0.25, 0.5, 1.0, 2.0, and 5.0 mL of the standard stock solution into six 10 mL amber volumetric flasks, and dilute to the mark with anhydrous ethanol to obtain a series of standard working solutions with concentrations of 0.0125, 0.025, 0.05, 0.10, 0.20, and 0.50. After HPLC analysis under the established chromatographic conditions, a standard curve was constructed with peak area as the ordinate (Y) and mass concentration as the abscissa (X). Figure 1 ).

[0052] The degradation rate of lutein was determined by high performance liquid chromatography. Four strains with lutein degradation ability were obtained from 198 culturable bacteria. Among them, strain YHS-1 showed the highest degradation rate of lutein, at 21.35% (see [link to article]). Figure 2 The degradation rate reached its peak after 48 hours of fermentation, and then began to decline around 60 hours. (See...) Figure 3 ).

[0053] The colonies of strain yhs-1 are round, smooth, light white, and have neat edges (see...). Figure 4 ).

[0054] 2. Molecular biological identification of strain YHS-1

[0055] Colony PCR was performed using universal primers 27F and 1492R for 16S rDNA. The PCR products were directly sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The obtained 16S rDNA gene nucleotide sequences were entered into the GenBank database for BLAST alignment. Sequences of strains with high 16S rDNA gene sequence similarity were obtained, and multiple sequence alignment was performed using MEGA X. A phylogenetic tree was constructed using Neighbor-joining. Figure 5 As shown, strain yhs-1 shares 98% similarity with Paenibacillus dendritiformis, thus identifying strain yhs-1 as a dendritic Bacillus. Dendritic Bacillus is widely found in plant leaves and is a potentially beneficial bacterium.

[0056] The strain yhs-1 is classified and named Paenibacillus dendritiformis yhs-1. It has been deposited at the China Center for Type Culture Collection (CCTCC) on October 25, 2023, with accession number CCTCC NO: M 20232033.

[0057] 3. Optimization of culture medium for strain YHS-1

[0058] With the inoculum size of YHS-1 fixed at 2% (V / V), rotation speed at 180 r / min, temperature at 37℃, and liquid volume at 100 mL / 250 mL, the optimal addition amounts of glucose (5, 10, 15, 20, 25 g / L), peptone (5, 10, 15, 20, 25 g / L), and MgSO4 (0.1, 0.2, 0.3, 0.4, 0.5 g / L) in the basal fermentation medium were determined through single-factor experiments. Then, a three-factor, three-level Box-Behnken response surface methodology experiment was designed to further optimize the results.

[0059] The results of the single-factor experiment are as follows:

[0060] The effect of glucose content on the degradation of lutein by strain YHS-1 is as follows: Figure 6 As shown, with increasing glucose content, the bacterial cell concentration increases, and the lutein degradation rate also increases. The degradation rate of lutein reaches its highest level when the glucose addition is 15 g / L. With further increases in glucose content, the degradation rate of lutein decreases, possibly because excessively high glucose concentrations inhibit the growth and metabolism of the bacterial strain, thus reducing its ability to degrade lutein.

[0061] The effect of peptone content on the degradation of lutein by strain YHS-1 is as follows: Figure 7 As shown, when the amount of peptone added was 15 g / L, the degradation rate and cell concentration of lutein reached the highest level, and then the degradation rate and cell concentration decreased with the increase of peptone content.

[0062] The effect of MgSO4 addition on the degradation of lutein by strain YHS-1 is as follows: Figure 8 As shown, the degradation rate of lutein initially increased and then decreased with increasing MgSO4 addition. Both the degradation rate and cell concentration reached their highest levels when the MgSO4 addition was 0.3 g / L. Similar to the effects of glucose and peptone on the degradation of lutein by strain yhs-1, the highest degradation rate was observed at the highest cell concentration, indicating that under these fermentation conditions, cell concentration has the most direct impact on lutein degradation.

[0063] Response surface methodology optimization design and results:

[0064] Based on the single-factor experiments, a three-factor, three-level design was constructed with glucose (A), peptone (B), and MgSO4 (C) as independent variables and the degradation rate of lutein as the response value (Table 1). The experimental results are shown in Table 2. Multiple regression analysis was performed on the experimental results using Design-ExpertV13 software, yielding the quadratic regression equation of lutein degradation rate on each factor: Y = 27.56 + 0.75A + 2.24B + 1.21C + 0.625AB + 0.525AC + 0.1BC - 2.21A² - 2.83B² - 0.98C². Further analysis of variance was performed, and the results are shown in Table 3.

[0065] Table 1. Box-Behnken Experimental Factors and Levels Design

[0066]

[0067] Table 2 Box-Behnken Experimental Design and Results

[0068]

[0069] Table 3. Analysis of Variance for Regression Models

[0070]

[0071] Note: "**" indicates extremely significant (P<0.01), "*" indicates significant (P<0.01). <P<0.05),P> 0.05 indicates that it is not significant.

[0072] Table 3 shows that the regression model P < 0.01 indicates that the model is highly significant, while the lack-of-fit term P = 0.2127 > 0.05 is not significant, indicating that the model fits the experimental conditions well. Regression coefficient R0 2 =0.9868, indicating a good fit of the regression equation and a good reflection of the relationship between lutein degradation rate and the glucose, peptone, and MgSO4 in the fermentation medium. Table 3 also shows that the linear and quadratic terms have highly significant effects on the results (P<0.01), while the interaction terms AC and BC have no significant effect, and the exchange term AB has a significant effect. From the F-values, the influence of each factor on the lutein degradation rate is: B>C>A. That is, peptone in the fermentation medium > MgSO4 > glucose.

[0073] The response surface and contour lines of the interaction between glucose, peptone, and MgSO4 on the degradation rate of lutein are shown in the figure. Figure 9As shown, the response surface of AB has a steeper slope and faster color change, and the contour lines are elliptical and dense, indicating that the interaction between AB has a significant effect (P<0.05); conversely, the interaction between AC and BC is not significant, which is consistent with the results of the analysis of variance.

[0074] The optimal fermentation medium conditions for lutein degradation, obtained from the model, were: glucose 15.958 g / L, peptone 16.330 g / L, and MgSO4 0.336 g / L. Under these conditions, the degradation rate of lutein was 28.62%. For ease of practical operation, the fermentation medium conditions were set to glucose 16 g / L, peptone 16 g / L, and MgSO4 0.3 g / L. Three parallel experiments were conducted under these conditions, and the results are as follows: Figure 10 As shown, the degradation rate of lutein was 26.4% after 48 h of fermentation, which deviated from the theoretical value by 7.7%.

[0075] 4. Identification of lutein degradation products

[0076] The fermentation products of strain yhs-1 were analyzed by GC-MS, and the differential substances with and without lutein addition were obtained (Table 4), including 1 aldehyde, 6 ethers, 5 esters, 4 alkanes, and 2 alcohols. Among them, octadecenal (C 18 H 34 O) and phytol (C 20 H 40 O) and other substances are typical flavor compounds. The production of these differentially expressed substances is a result of the degradation and transformation of lutein by strain yhs-1. Compared with the resting cell method for determining the products of lutein degradation, the method used in this study is closer to the actual application of the strain, because fruits, vegetables and tobacco contain a large number of other nutrients besides lutein, which can meet the nutritional needs of strain yhs-1. Under these circumstances, the degradation and transformation of lutein involves a more complex metabolic pathway.

[0077] Table 4. Differences in fermentation production with and without lutein in the culture medium.

[0078]

[0079] 5. Application of Bacillus dendriticis YHS-1 in tobacco fermentation

[0080] Preparation of bacterial suspension: *Bacillus dendriticis* yhs-1 was streaked onto LB solid medium and incubated at 37°C for 24 h. Single colonies were then picked and transferred to LB liquid medium, and cultured at 37°C with shaking at 180 r / min until the OD of the bacterial suspension reached approximately 600 to 1 to obtain a seed culture. The culture was then expanded at a 2% (V / V) inoculation ratio to obtain sufficient bacterial suspension. The suspension was centrifuged at 4°C and 6000 r / min for 5 min, and the supernatant was discarded to obtain a bacterial pellet. The pellet was resuspended in sterile PBS and washed once to adjust the bacterial concentration to 5 × 10⁻⁶. 8 CFU / mL, obtain a bacterial suspension for later use.

[0081] Tobacco leaf fermentation: The bacterial suspension was sprayed onto the surface of tobacco shreds (the tobacco leaves were cut into shreds, 2 mm wide) at a mass-to-volume ratio of 50:1 (Kg:L). Fermentation was carried out at a temperature of 37 ℃ and a humidity of 55% for 48 h. After fermentation, the lutein content of the tobacco leaves before and after fermentation was measured and the tobacco leaves were evaluated.

[0082] The sensory quality evaluation standards are as follows: For tobacco leaves before and after fermentation, in accordance with the standard requirements of tobacco industry YC / T497-2014, the evaluation team organized smokers to evaluate the changes in sensory quality of tobacco leaves before and after fermentation based on 10 indicators: aroma quality, aroma quantity, penetration, concentration, off-flavors, irritation, sweetness, dryness, strength, and aftertaste. Each indicator has a full score of 10 points. The total sensory quality evaluation score is calculated based on the sum of the scores of the evaluation indicators. The results are shown in Table 5.

[0083]

[0084] As shown in the table above, after fermentation with strain YHS-1, the lutein content in tobacco leaves decreased from 210.5 μg / g before fermentation to 160.7 μg / g, with a degradation rate of 23.66%. Sensory evaluation results showed that compared with unfermented tobacco leaves, fermented tobacco leaves had higher sensory scores, reduced off-flavors, lower irritation, a purer and more comfortable aftertaste, and increased aroma.

[0085] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A tree-like Paenibacillus characterized by, The taxonomic name of the Paenibacillus dendritiformis is Paenibacillus dendritiformis yhs-1, which has been preserved in the China Center for Type Culture Collection (CCTCC) on October 25, 2023, and the preservation number is CCTCC NO: M 20232033.

2. A bacterial suspension, characterized in that, The Paenibacillus dendritiformis according to claim 1.

3. The bacterial suspension according to claim 2, characterized in that, The bacterial suspension is prepared by the following method: Single colony was picked to LB liquid medium, and cultured by shaking to OD 600 The seed liquid was obtained by culturing to OD 0.8-1.2, and the bacteria liquid was obtained by culturing to 1.8-2.2% inoculation ratio. The bacteria liquid was centrifuged, and the supernatant was discarded to obtain the bacteria precipitate. The bacteria precipitate was resuspended and washed with sterile PBS to obtain the bacteria suspension.

4. The Paenibacillus dendritiformis according to claim 1 and / or the bacterial suspension according to claim 2 are used for degrading lutein.

5. The Paenibacillus dendritiformis according to claim 1 and / or the bacterial suspension according to claim 2 are used for enhancing the flavor of tobacco.

6. Use of the Paenibacillus chondritus of claim 1 and / or the bacterial suspension of claim 2 in the fermentation of tobacco leaves, characterized in that, The bacterial suspension of the Paenibacillus dendritiformis is sprayed on the surface of tobacco shreds for fermentation.

7. The Paenibacillus dendritiformis according to claim 1 and / or the bacterial suspension according to claim 2 are used for improving the quality of tobacco leaves.

8. Use according to claim 6, characterized in that, The fermentation condition is 45-60 h of fermentation at a temperature of 32-40 ℃ and a humidity of 45-65%.

9. Use according to claim 6, characterized in that, The volume-to-mass ratio of the bacterial suspension to tobacco shreds is 1:45-55.

10. Use according to claim 6, characterized in that, The concentration of the bacterial suspension is 5 x 10 7 ~ 5 x 10 10 CFU / mL.

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