Bacillus aerobacter and application thereof in degradation of protein

Abstract

The invention belongs to the field of microorganisms and biology, and provides bacillus aerosus and application thereof in degradation of protein, the strain is preserved in China General Microbiological Culture Collection Center on November 22, 2023, the preservation number is CGMCC No. 29096, and the preservation number is CGMCC No. 29096. The address of the preservation unit is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101. The strain is applied to cigar tobacco leaves, the protein content in the cigar tobacco leaf fermentation process can be effectively reduced, the aroma of the tobacco leaves can be enhanced, sensory indexes such as the aroma quality, the aroma amount, the aftertaste and the pleasure of the tobacco leaves are improved, and a research thought is provided for development of cigar products with novel characteristic aroma and cigar industrial microbial preparations.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biotechnology, and in particular relates to an air bacillus and application thereof in degrading protein. 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 made entirely from cigar tobacco leaves, which must undergo a period of fermentation or aging to significantly improve their quality and processing properties, meeting the requirements of industrial cigar rolling. Currently, cigar tobacco leaf fermentation technology remains the most challenging core technology in cigar raw material production.

[0004] Protein is one of the main nutrients in tobacco leaves. The protein content in tobacco leaves is closely related to the quality of tobacco leaves. Screening and using functional microorganisms that can degrade protein will help reduce the white matter content in tobacco leaves, reduce irritation, and improve the quality of cigar tobacco leaves.

[0005] To this end, some studies have reported the use of the Bacillus subtilis XP strain for degrading protein in tobacco products, and others have reported the use of the Bacillus pumilus SMXP-03 strain for degrading protein in tobacco leaves. However, for cigars, these methods fail to achieve a consistent degradation rate while also enhancing the aroma and flavor of the cigars. Summary of the Invention

[0006] To solve the above problems, the present invention provides an airborne Bacillus and its application in protein degradation. The strain of the present invention can enhance the aroma of cigars while ensuring the degradation rate, enriching the flavor of cigars.

[0007] 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 Bacillus aerius The strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on November 22, 2023, with the deposit number: CGMCC No. 29096. The deposit unit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0008] The second aspect of the present invention provides the above-mentioned air bacillus Bacillus aerius Application in degrading protein and / or improving sensory quality of tobacco leaves.

[0009] The third aspect of the present invention provides a bacterial agent for degrading tobacco leaf protein, the bacterial agent comprising: the above-mentioned Bacillus aeruginosa Bacillus aerius .

[0010] A fourth aspect of the present invention provides a method for preparing a bacterial agent for degrading tobacco leaf protein, comprising: The aerobic Bacillus is inoculated into a culture medium at an inoculation rate of ≥1%, and cultured at 35° C.-40° C. and 200 rpm-240 rpm for 24 h-48 h to obtain the product.

[0011] A fifth aspect of the present invention provides a method for degrading protein in tobacco leaves, comprising: Spray the above-mentioned bacterial agent onto the surface of tobacco leaves with an inoculation amount of ≥20%, and ferment to obtain the product.

[0012] Beneficial effects of the present invention (1) The present invention provides an airborne Bacillus, which can be applied to cigar tobacco leaves and effectively reduce the protein content during the fermentation process of cigar tobacco leaves, enhance the aroma of tobacco leaves, and improve sensory indicators such as aroma quality, aroma volume, aftertaste and pleasant feeling of tobacco leaves, providing 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 enhance the aroma of cigars while ensuring the degradation rate, enriching the flavor of cigars. The fermentation method is simple, 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 This is the test result of protein content in tobacco leaves; Figure 2 This is a sensory evaluation chart. 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] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in accordance with conventional methods in the art or according to product specifications. Similarly, unless otherwise specified, the test methods of the present invention are also tested in accordance with conventional methods in the art or the common methods or standards in the industry. In addition, any methods and materials similar to or equivalent to the described contents can be applied to the inventive method. The preferred embodiments and materials described herein are for demonstration purposes only.

[0018] The first aspect of the present invention provides a strain of Bacillus aeruginosa Bacillus aerius The strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on November 22, 2023, with the deposit number: CGMCC No. 29096. The deposit unit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0019] The second aspect of the present invention provides the above-mentioned air bacillus Bacillus aerius Application in degrading protein and / or improving sensory quality of tobacco leaves.

[0020] In some embodiments, the protein is a protein from tobacco leaves.

[0021] The present invention does not impose any particular limitation on the specific tobacco variety to be processed. Preferably, in some embodiments, the tobacco leaves are cigar tobacco leaves to obtain better degradation effect and aroma improvement effect.

[0022] In some embodiments, the degradation method is fermentation. Fermentation can effectively reduce the protein content of cigar tobacco during fermentation, enhance the aroma of the tobacco, and improve sensory indicators such as aroma quality, aroma volume, aftertaste, and pleasant feeling.

[0023] The third aspect of the present invention provides a bacterial agent for degrading tobacco leaf protein, the bacterial agent comprising: the above-mentioned Bacillus aeruginosa Bacillus aerius .

[0024] 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 Bacillus aerius The concentration is ≥1×10 8 CFU / mL to effectively degrade protein, enhance the aroma of tobacco leaves, and improve sensory indicators such as tobacco aroma quality, aroma volume, aftertaste and pleasure.

[0025] A fourth aspect of the present invention provides a method for preparing a bacterial agent for degrading tobacco leaf protein, comprising: The aerobic Bacillus is inoculated into a culture medium at an inoculation rate of ≥1%, and cultured at 35° C.-40° C. and 200 rpm-240 rpm for 24 h-48 h to obtain the product.

[0026] A fifth aspect of the present invention provides a method for degrading protein in tobacco leaves, comprising: Spray the above-mentioned bacterial agent onto the surface of tobacco leaves with an inoculation amount of ≥20%, and ferment to obtain the product.

[0027] Moisturizing tobacco leaves can balance moisture, increase leaf temperature, increase toughness, reduce breakage, and improve tobacco smoking quality. Therefore, in some embodiments, tobacco leaves sprayed with microbial inoculants are allowed to retard under constant temperature and humidity conditions for 24-32 hours before fermentation to further improve tobacco leaf quality.

[0028] Fermentation conditions can affect the degradation of carotenoids and the improvement of aroma. Therefore, the present invention has studied the fermentation conditions. In some embodiments, the fermentation conditions are 45-48°C and the humidity is 90%-92% to better degrade protein, enhance the aroma of tobacco leaves, and improve sensory indicators such as tobacco aroma quality, aroma volume, aftertaste and pleasure.

[0029] 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.

[0030] In the following examples, all raw materials are commercially available products.

[0031] The present invention screened out a protein-degrading strain from cigar 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, and the 16S rRNA sequence was compared using BLAST on NCBI. The homology was 98.9%, and the strain was identified as Bacillus aeruginosa ( Bacillus aerius ). It grows in beef extract peptone agar medium, and its colony characteristics are: large colony diameter, milky white colony, and irregular shape.

[0032] Example 1 Application of Aerobacillus in cigar tobacco fermentation.

[0033] The specific steps are as follows: (1) Air Bacillus ( Bacillus aerius) was inoculated into beef extract peptone liquid culture medium at a 1% inoculation rate and cultured at 37°C and 220 rpm for 48 h to obtain a fermentation broth. At this time, the air Bacillus ( Bacillus aerius ) concentration is at least 1×10 8 CFU / mL. Use a spray bottle to evenly spray the preparation on the surface of Hainan cigar tobacco leaves at a 20% inoculum rate.

[0034] (2) Place the tobacco leaves sprayed with the fermentation liquid preparation in step (1) in a ziplock bag, mark it as the inoculation group, and allow it to recover moisture at room temperature in a constant temperature and humidity incubator for 24 hours.

[0035] (3) The specific conditions for fermentation culture are: set the temperature of the constant temperature and humidity incubator to 45℃ and the humidity to 90%, ferment for 5 days, and turn the tobacco leaves over every day.

[0036] (4) After fermentation, store at -20°C.

[0037] Comparative Example 1 The specific steps are as follows: (1) Use a spray bottle to evenly spray sterile water (commercially available) on the surface of Hainan cigar tobacco leaves at a 20% inoculum volume and mark it as CK.

[0038] (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.

[0039] (3) The specific conditions for fermentation culture are: set the temperature of the constant temperature and humidity incubator to 45℃ and the humidity to 90%, ferment for 5 days, and turn the tobacco leaves over every day.

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

[0041] Experimental Example 1 Component Analysis The protein content of the fermented cigar tobacco leaves in Example 1 and Comparative Example 1 was analyzed in the following steps: The protein content of post-fermentation tobacco leaves was determined using the Coomassie Brilliant Blue method. After fermentation, the leaves were air-dried. 0.2 g of sample was accurately weighed in a mortar and pestle, added to 5 mL of phosphate buffer (pH 7.0), and ground into a homogenate in an ice bath. The mixture was centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was poured into a 10 mL volumetric flask, and the residue was resuspended in 2 mL of phosphate buffer. The mixture was then centrifuged under the same conditions, and the supernatants were combined and mixed. Protein content was determined using a protein quantification (TP) kit.

[0042] It has been determined that Figure 1As shown, the protein content of cigar tobacco leaves without strain fermentation was 19.30±0.29 mg / g, and the protein content of cigar tobacco leaves fermented with strain was 10.63±1.36 mg / g. Compared with the control group, the protein content decreased significantly by 44.92%.

[0043] Experimental Example 2 Sensory Evaluation The sensory evaluation is scored on a 100-point scale, with a maximum score of 100 points. The items are aroma volume (15), aroma quality (10), pleasantness (5), harmony (5), smoke concentration (5), miscellaneous odors (20), irritation (10), aftertaste (15), color (5), burnability (5), and gray (5). Each item is scored in units of 0.5 points. When scoring the aroma characteristics, smoke characteristics, taste characteristics, and appearance characteristics, each item will be scored accordingly if the quality meets the requirements of the corresponding item.

[0044] like Figure 2 As shown in the results, the total sensory evaluation score of cigar tobacco leaves not fermented with the strain was 54, while the total sensory evaluation score of cigar tobacco leaves fermented with the strain was 77.5. Compared with the control group, the total sensory evaluation score of the strain-fermented group was significantly higher. Among them, except for a slight decrease in combustibility, all other scores showed improvements, with the aroma quality, aroma volume, and pleasantness scores increasing significantly.

[0045] 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 are intended to be 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 of Microorganisms on November 22, 2023, with the deposit number: CGMCC No. 29096. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

2. Air Bacillus as claimed in claim 1 bacillus aerius Application in degrading protein and / or improving sensory quality of tobacco leaves.

3. The use according to claim 2, characterized in that Protein is the protein 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. A bacterial agent for degrading tobacco leaf protein, characterized in that: The bacterial agent includes: the air bacillus according to claim 1 bacillus aerius .

7. The tobacco leaf protein-degrading bacterial agent according to claim 6, wherein: Air Bacillus bacillus aerius The concentration is ≥1×10 8 CFU / mL.

8. A method for preparing a bacterial agent for degrading tobacco leaf protein, characterized in that: include: The aerobic Bacillus is inoculated into a culture medium at an inoculation rate of ≥1%, and cultured at 35° C.-40° C. and 200 rpm-240 rpm for 24 h-48 h to obtain the product.

9. A method for degrading protein in tobacco leaves, characterized in that: include: The bacterial agent according to claim 6 or 7 is sprayed onto the surface of tobacco leaves with an inoculation amount of ≥20%, and fermented to obtain the product.

10. The method for degrading protein in tobacco leaves according to claim 9, wherein: The tobacco leaves sprayed with the microbial agent are allowed to recover moisture for 24-32 hours under constant temperature and humidity conditions before fermentation. The fermentation conditions are 45-48° C. and 90%-92% humidity.

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