Bacillus aerobacter and application thereof in degradation of carotenoid

Abstract

The invention relates to the technical field of microorganisms and biology, in particular to bacillus aerosus and application thereof in degradation of carotenoid. The invention provides an aerobacillussp strain and application of the aerobacillussp strain in degradation of carotenoids in tobacco leaves, and the aerobacillussp strain can effectively promote degradation of the carotenoids in the tobacco leaves and increase the content of substances such as beta-ionone, geranyl acetone and farnesyl acetone in the tobacco leaves.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biotechnology, and particularly relates to an air bacillus and application thereof in degrading carotenoids. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Cigars are tobacco products with a unique aroma. To further improve the aroma and quality of tobacco leaves, extensive research has been conducted on the screening and application of functional strains to enhance the aroma and quality of low-grade tobacco leaves. For example, incorporating β-carotene degradation products into cigarettes can improve the aroma quality and volume of tobacco leaves, reduce off-flavors and irritation, and improve the aftertaste. However, limited research has been conducted on the screening and application of strains that degrade carotenoids in tobacco leaves.

[0004] A study has disclosed a method for improving tobacco leaf quality, in which Bacillus subtilis with the ability to degrade carotenoids was isolated from flue-cured tobacco. A study has disclosed a method for improving tobacco leaf quality by synergistic treatment with bacterial enzymes, in which Bacillus pumilus ( Bacillus pumilus ) and carotenoid 9,10′ dioxygenase synergistically treat tobacco leaves. However, for cigar tobacco, the industry still urgently needs to develop strains that can effectively degrade carotenoids, enhance tobacco flavor and aroma quality, and reduce off-flavors. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides an airborne Bacillus sp. and also provides the use of the strain in degrading carotenoids in tobacco leaves, which can effectively promote the degradation of carotenoids in tobacco leaves and increase the content of substances such as β-ionone, geranylacetone, and farnesylacetone in tobacco leaves.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a strain of Bacillus aeruginosa aeruginosa The strain was deposited in the General Microbiology Center of China Culture Collection Administration on November 22, 2023, with the deposit number: CGMCC No. 29096.

[0007] The second aspect of the present invention provides the above-mentioned air bacillus aeruginosa Application in degradation of carotenoids.

[0008] The third aspect of the present invention provides the above-mentioned air bacillus aeruginosaIts application in increasing the content of geranyl acetone, farnesyl acetone and β-ionone in tobacco degradation products.

[0009] The fourth aspect of the present invention provides a bacterial agent, which comprises: the above-mentioned Bacillus aeruginosa aeruginosa .

[0010] A fifth aspect of the present invention provides a method for preparing a microbial agent, comprising: The aerobic Bacillus is inoculated into a beef extract peptone liquid culture medium at an inoculation amount of ≥1%, and cultured at 35° C.-40° C. and 200 rpm-240 rpm for 24 h-48 h to obtain the culture medium.

[0011] A sixth aspect of the present invention provides a method for degrading carotenoids in tobacco leaves, comprising: The above-mentioned air Bacillus aeruginosa Or the above-mentioned bacterial agent is mixed evenly with tobacco leaves and fermented to obtain the product; Among them, Bacillus spp. aeruginosa The concentration is ≥1×10 8 CFU / mL.

[0012] Beneficial effects of the present invention (1) The present invention provides an airborne Bacillus, which is applied to cigar tobacco leaves and can effectively reduce the content of carotenoids during the fermentation process of cigar tobacco leaves, increase the content of substances such as β-ionone, geranylacetone, and farnesylacetone in tobacco leaves, enhance the bean aroma, woody aroma, and nutty aroma of tobacco leaves, improve the aroma quality and aroma quantity of tobacco leaves, reduce sensory indicators such as miscellaneous odor and irritation, and provide research ideas for the development of new characteristic aroma cigar products and cigar industrial microbial preparations.

[0013] (2) The strain of the present invention can effectively degrade carotenoids, increase the aroma and flavor quality of tobacco leaves, and reduce the smell of other smells. It is highly practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0015] Figure 1 The following is a picture of the colony of the strain in beef extract peptone agar medium. DETAILED DESCRIPTION

[0016] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0017] The present invention provides a strain of air bacillus aeruginosa The strain was deposited in the General Microbiology Center of China Culture Collection Administration on November 22, 2023, with the deposit number: CGMCC No. 29096.

[0018] The inventors of this application screened out a carotenoid-degrading strain from tobacco leaves, and used bacterial universal primers 27F and 1492R to perform colony PCR on the screened carotenoid-degrading strain. The obtained PCR product was sent to Qingdao Weilai Company for sequencing. After BLAST was used to perform 16S rRNA sequence comparison on NCBI, the homology was 98.9%, and the strain was identified as Bacillus aeruginosa ( Bacillus aereus ). It grows in beef extract peptone agar medium, and its colony characteristics are: large colony diameter, milky white colony, and irregular shape.

[0019] Culture collection information Preservation time: November 22, 2023 Depository: China General Microbiology Center, China Culture Collection Administration Committee, Deposit number: CGMCC No. 29096, Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101 Classification name: Air Bacillus aeruginosa .

[0020] The present invention also provides the above-mentioned air bacillus aeruginosa Application in degradation of carotenoids.

[0021] In some embodiments, the carotenoids are carotenoids in tobacco leaves. The present invention is not limited to the degradation of carotenoids in tobacco leaves, but can also be used to degrade carotenoids in other plants.

[0022] The present invention does not impose any particular limitation on the specific tobacco variety to be processed. Preferably, the tobacco leaves are cigar tobacco leaves to obtain better degradation effect and aroma improvement effect.

[0023] In some embodiments, the degradation method is fermentation, which can effectively degrade carotenoids in tobacco leaves and increase the content of substances such as β-ionone, geranylacetone, and farnesylacetone in tobacco leaves.

[0024] The present invention also provides a method for degrading carotenoids in tobacco leaves, comprising: The above-mentioned air Bacillus aeruginosa Or the above-mentioned bacterial agent is mixed evenly with tobacco leaves and fermented to obtain the product; Among them, Bacillus spp. aeruginosa The concentration is ≥1×10 8 CFU / mL.

[0025] Fermentation conditions affect the degradation effect of carotenoids and the improvement effect of aroma. Therefore, the present invention studies the fermentation conditions. In some embodiments, the fermentation conditions are 35°C-39°C and 68%-72% humidity for 5-10 days to obtain better carotenoid degradation effect and aroma improvement effect.

[0026] The amount of air bacillus directly affects the fermentation cycle and the time of product formation. Therefore, the present invention has studied the amount of air bacillus. In some embodiments, the amount of air bacillus is aeruginosa The dosage ratio of tobacco leaves is (40-50) mL: 200 g to obtain better fermentation effect.

[0027] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0028] Example 1 Isolation, screening and identification of Bacillus aeruginosa strains.

[0029] 1. Isolation of strains The specific steps are as follows: Ten grams of tobacco leaf sample provided by Shandong China Tobacco Industry Co., Ltd. was ground with liquid nitrogen (to promote the release of endophytes). The ground powder was added to 90 mL of beef extract peptone medium containing 20 g / L tobacco leaf. The culture was shaken at 37°C and 220 rpm for 48 h to obtain a culture medium. 2. Screening of strains The specific steps are as follows: (1) The culture medium obtained above was diluted to 10 -7 , spread on the screening medium and culture for 24 hours, pick the strains with transparent rings for rescreening. Then, weigh 10 mg of tobacco leaves in a 250 mL triangular flask, add 100 mL of sterile water and soak for 24 hours, filter, take 2 mL of filtrate and add it to the enrichment medium, culture at 37°C and 150 r / min for 2 days to obtain the bacterial source. Take an appropriate amount of bacterial source for gradient dilution, select 10 -3 , 10 -4 , 10 -5Three concentration gradients were plated on the separation medium, with 3 plates plated in parallel for each concentration gradient. The plated plates were cultured in an incubator at 37°C for 2-3 days, and single colonies with good morphology and transparent circles were selected and repeatedly streaked for separation and purification.

[0030] (2) Single colonies with obvious transparent zones were selected and inoculated into 100 mL of fermentation medium at 28°C and 150 r / min for 2 days. The strains with the ability to degrade carotenoids were further screened by observing the color of the shake flasks and detecting the degradation products by GC-MS analysis.

[0031] 3. Identification of strains The specific steps are as follows: (1) The 16S rRNA gene of the obtained strain was amplified using the universal bacterial primers 27F (5′-AGAGTTTGATCMTGG CTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).

[0032] (2) PCR was performed using 30 μL of reaction mixture and Taq DNA Polymerase (Takara) was selected. The PCR conditions were pre-denaturation at 95°C for 5 min, followed by 35 cycles of amplification at 95°C for 30 s, 58°C for 30 s, and 72°C for 1.5 min to obtain the PCR product.

[0033] (3) The PCR product obtained in step (2) was analyzed by 1% agarose gel electrophoresis and sent to Qingdao Weilai Company for sequencing.

[0034] (4) The sequence was compared with the previously published bacterial 16S rRNA sequence in the NCBI database, and the homology was 98.9%. The bacterial strain in beef extract peptone solid medium had the following colony characteristics: the colony diameter was large, the colony was milky white, and the shape was irregular, such as Figure 1 shown.

[0035] (5) The strain was identified as Bacillus aereus , belongs to the genus Bacillus, and is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) with the number: 29096.

[0036] Example 2 Application of Aerobacillus in cigar tobacco fermentation.

[0037] The specific steps are as follows: (1) The strain was inoculated into beef extract peptone liquid medium at a 1% inoculum and cultured at 37°C and 220 rpm for 48 h to obtain a fermentation broth. 10 mL of the fermentation broth was added to sterile water to make a final volume of 50 mL. The mixture was mixed to obtain a preparation. At this time, the concentration of Bacillus aeruginosa in the preparation was at least 1×10 8 CFU / mL. Use a spray bottle to evenly spray the preparation on the surface of 250g Hainan cigar tobacco leaves.

[0038] (2) The tobacco leaves sprayed with the fermentation liquid preparation in step (1) are placed in a ziplock bag and allowed to recover moisture at room temperature in a constant temperature and humidity incubator for 24 hours.

[0039] (3) Fermentation conditions: The temperature of the constant temperature and humidity incubator was set at 37°C and the humidity was set at 70%. The fermentation was carried out for 5 days, and the tobacco leaves were turned over every day.

[0040] (4) After fermentation, store at -20°C.

[0041] Comparative Example 1 Blank control group The specific steps are as follows: (1) Use a spray bottle to evenly spray 46 mL of sterile water on the surface of Hainan cigar tobacco leaves and mark them as CK.

[0042] (2) Place the tobacco leaves sprayed with sterile water in step (1) in a ziplock bag and allow to humidify at room temperature in a constant temperature and humidity incubator for 24 hours.

[0043] (3) Fermentation conditions: The temperature of the constant temperature and humidity incubator was set at 37°C and the humidity was set at 70%. The fermentation was carried out for 5 days, and the tobacco leaves were turned over every day.

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

[0045] Experimental Example 1 Component Analysis The components of the fermented cigar tobacco leaves in Example 2 and Comparative Example 1 were analyzed in the following steps: (1) Gas chromatography-mass spectrometry (GC-MS) was used to determine the content of carotenoid degradation products in fermented tobacco leaves. The fermented tobacco leaves were dried at 40°C and ground into powder. 5 g of tobacco leaf powder was placed in a headspace bottle and 100 μg·μL of phenylethyl ester was added as an internal standard. -1 The helium flow rate was 1 mL·min -1 , the oven temperature was fixed at 40 °C for 2 min, then increased to 250 °C at 15 °C / min and maintained for 5 min, the ion source temperature was 210 °C, and the transfer line temperature was 280 °C.

[0046] (2) The computer spectral library WILEY 8.0 and the NIST14 library were used to compare and analyze the compounds. Carotenoid degradation products were selected for analysis, and the content was calculated using the absolute quantification method. The results are shown in Table 1 below.

[0047] Table 1. Comparison of degradation products

[0048] Table 1 shows that the content of carotenoid degradation products in tobacco leaves fermented with the strain increased by 2.08-fold, indicating that strain A1 significantly promoted carotenoid degradation in cigar tobacco leaves. Compared with CK, strain A1 increased β-ionone content by 4.16-fold, geranylacetone content by 4.85-fold, and farnesylacetone content by 3.51-fold. This increase in carotenoid degradation products plays a significant role in enhancing tobacco leaf aroma, improving aroma quality, and reducing off-flavors.

[0049] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A strain of airborne Bacillus bacillus aerius , characterized in that, This strain was deposited in the General Microbiology Center of China Culture Collection Administration on November 22, 2023, with the deposit number: CGMCC No. 29096.

2. Air Bacillus as claimed in claim 1 bacillus aerius Application in degradation of carotenoids.

3. The use according to claim 2, characterized in that Carotenoids are carotenoids found in tobacco leaves.

4. The use according to claim 3, characterized in that The tobacco leaves are cigar tobacco leaves.

5. The use according to claim 2, characterized in that The degradation method is fermentation.

6. Air Bacillus as claimed in claim 1 bacillus aerius Its application in increasing the content of geranyl acetone, farnesyl acetone and β-ionone in tobacco degradation products.

7. A bacterial agent, characterized in that The bacterial agent includes: the air bacillus according to claim 1 bacillus aerius .

8. A method for preparing a microbial agent, characterized in that: include: The aerobic Bacillus is inoculated into a beef extract peptone liquid culture medium at an inoculation amount of ≥1%, and cultured at 35° C.-40° C. and 200 rpm-240 rpm for 24 h-48 h to obtain the culture medium.

9. A method for degrading carotenoids in tobacco leaves, characterized in that: include: The airborne Bacillus according to claim 1 bacillus aerius Or the bacterial agent according to claim 7 is mixed evenly with tobacco leaves and fermented to obtain; Among them, Bacillus spp. bacillus aerius The concentration is ≥1×10 8 CFU / mL.

10. The method for degrading carotenoids in tobacco leaves according to claim 9, wherein: The fermentation conditions are as follows: a temperature of 35° C.-39° C. and a humidity of 68%-72% for 5-10 days. or, Bacillus aeruginosa bacillus aerius The dosage ratio with tobacco leaves is (40-50) mL: 200 g.

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