Bacillus subtilis and its application in degrading tannins from Zanthoxylum bungeanum leaves

Abstract

The invention discloses a bacillus subtilis and an application thereof in degrading tannin in zanthoxylum leaves. The classification nomenclature of the bacillus subtilis is Bacillus subtilis w‑4, which has been registered and preserved in the China Center for Type Culture Collection on April 29, 2024, and the preservation number is CCTCC NO: M 2024804. The bacillus subtilis can ferment and degrade tannin in zanthoxylum leaves, with a degradation rate of more than 90%, thereby improving the palatability of zanthoxylum leaves, providing a basis for applying zanthoxylum leaves to animal feed, and adding an appropriate amount of fermented zanthoxylum leaves to animal feed can reduce the demand for imported raw materials such as corn and soybeans in animal feed, promote animal digestion, and promote the resource utilization of zanthoxylum leaves, which are byproducts in the production process of zanthoxylum.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to a Bacillus subtilis and application thereof in degrading tannin in Zanthoxylum bungeanum leaves. Background Art

[0002] The fruit of Sichuan pepper is small in size. In the process of collecting Sichuan pepper raw materials, Sichuan pepper fruits are often picked together with Sichuan pepper branches and leaves. The raw materials for making Sichuan pepper in the food industry are mainly Sichuan pepper peels. In the process of Sichuan pepper production, a large number of by-products such as Sichuan pepper leaves, Sichuan pepper seeds and Sichuan pepper bark are produced. These by-products are often discarded or incinerated, which ultimately causes environmental pollution and waste of resources. Sichuan pepper leaves are rich in nutrients. Adding Sichuan pepper leaves to animal feed can reduce the content of corn, soybeans, etc. in animal feed and reduce the demand for raw materials such as corn and soybeans. However, Sichuan pepper leaves contain a large amount of cellulose and tannins. Cellulose will cause low digestibility after animal consumption, and tannins will affect the palatability of animals to feed and the digestion and absorption of nutrients in feed by animals, making animals feel uncomfortable after consumption. Therefore, studying how to degrade cellulose and tannins in Sichuan pepper leaves, improve their palatability, promote animal feeding, and improve feed utilization plays an important role in promoting the promotion and application of production by-products such as Sichuan pepper leaves. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] In order to achieve these purposes and other advantages of the present invention, a Bacillus subtilis is provided, whose classification nomenclature is Bacillus subtilis w-4, which has been registered and preserved in the China Center for Type Culture Collection on April 29, 2024, and the preservation number is CCTCC NO: M 2024804.

[0005] An application of the Bacillus subtilis as described above in degrading tannins from Zanthoxylum bungeanum leaves.

[0006] An application of the above-mentioned Bacillus subtilis in degrading Zanthoxylum bungeanum leaf tannins comprises the following steps:

[0007] Step 1: Activation of bacteria: Prepare activation culture medium plates, take out the stored Bacillus subtilis w-4 and thaw it at room temperature, streak the plates in a clean bench, and place them in an incubator for culture;

[0008] Step 2: Prepare seed solution: prepare seed culture medium, take out the activated culture medium plate after culturing for 10-14 hours, transfer it to the seed culture medium, and culture it in a constant temperature shaker;

[0009] Step 3: Determination of colony count: Using the viable cell counting method, take the seed solution and perform 10-fold serial dilutions, spread the final dilution on the activated culture medium, place it in an incubator for culture, and calculate the number of viable bacteria;

[0010] Step 4: Inoculation of bacteria, the method is as follows:

[0011] S41, mixing Zanthoxylum bungeanum leaf powder and distilled water, sterilizing at 121° C. for 20 min, taking out after cooling, obtaining a fermentation medium, and storing at room temperature;

[0012] S42. Operate in a clean bench, take the seed solution and centrifuge it at 1500-2500 rpm for 4-7 minutes, collect the bacteria, dilute it with the seed culture medium, and inoculate it into the fermentation medium, stir and mix, and then seal it and put it into the incubator for fermentation;

[0013] Step 5. Collect samples: After fermentation, take out the fermented pepper leaves, dry them, collect and seal them, and store them at room temperature away from light.

[0014] Preferably, in step 1, Bacillus subtilis w-4 is stored at -80°C; and the incubator temperature is 33-40°C.

[0015] Preferably, the activation culture medium comprises: 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl solid, 20 g / L agar, and is sterilized at 121° C. for 20 min.

[0016] Preferably, in step 2, the temperature of the constant temperature shaker is 33-40°C, the speed is 100-150 r / min, and the culture is carried out until the OD 600 =0.5~0.7.

[0017] Preferably, the seed culture medium is: 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl solid, 1000 mL distilled water, and sterilized at 121° C. for 20 min.

[0018] Preferably, in step three, the culture is carried out in a 33-40° C. incubator for 10-14 hours.

[0019] Preferably, in step 4, the mass volume ratio of Zanthoxylum bungeanum leaf powder and distilled water is 1 g: 1.0-2.0 mL; the mixture is sealed and placed in an incubator at 33-37° C. for fermentation for 3-5 days; the inoculation amount of the strain is 8×10 7 ~2×10 8 CFU / g; mass of Zanthoxylum bungeanum leaf powder: total volume of distilled water and seed culture medium is 1g: 1.5~2.5mL.

[0020] Preferably, in step 5, the drying is performed by placing the product in an oven at 60-70° C. for 20-28 hours.

[0021] In order to further improve the degradation rate of Zanthoxylum bungeanum leaf tannin by Bacillus subtilis w-4, the S41 is replaced by: taking 1000 mL of distilled water, adding 3-7 g of sucrose, 3-7 g of sorbitol, 0.05-0.2 g of cyclohexanehexol, 0.5-2 g of sodium acetate, 0.005-0.03 g of ZnSO4 and 2-5 g of diammonium hydrogen citrate, stirring evenly, adjusting the pH to 7.0-7.5, and putting it into a high-pressure homogenizer for secondary homogenization, that is, first homogenizing at 10-20° C. and 20-50 MPa for 4-6 min, and then homogenizing at 60-100° C. and 80-120 MPa for 4-6 min to obtain a mixed solution; mixing the Zanthoxylum bungeanum leaf powder and the mixed solution at a mass volume ratio of 1 g: 0.5-2 mL, sterilizing at 121° C. for 20 min, taking out after cooling, obtaining a fermentation medium, and storing it at room temperature.

[0022] The present invention at least includes the following beneficial effects: the present invention discloses a Bacillus subtilisw-4 with a preservation number of CCTCC NO: M2024804, which can degrade tannins in Zanthoxylum bungeanum leaves through fermentation, with a degradation rate of more than 90%, thereby improving the palatability of Zanthoxylum bungeanum leaves and providing a basis for applying Zanthoxylum bungeanum leaves to animal feed. Adding an appropriate amount of fermented Zanthoxylum bungeanum leaves to animal feed can reduce the demand for imported raw materials such as corn and soybeans in animal feed, promote animal digestion, and promote the resource utilization of Zanthoxylum bungeanum leaves, which are a by-product in the production process of Zanthoxylum bungeanum.

[0023] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the tannin standard curve of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0026] It should be understood that terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0027] In the following embodiments:

[0028] Activation medium: beef extract 3g / L, peptone 10g / L, NaCl solid 5g / L, agar 20g / L, sterilized at 121℃ for 20min;

[0029] Seed culture medium: 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl solid, 1000 mL distilled water, sterilize at 121°C for 20 min.

[0030] Example 1

[0031] Application of Bacillus subtilis in degrading Zanthoxylum bungeanum leaf tannins comprises the following steps:

[0032] Step 1: Activation of bacterial strains: Prepare activation culture medium plates, take out Bacillus subtilis w-4 with the deposit number of CCTCC NO: M2024804 stored at -80°C, thaw at room temperature, streak the plates in a clean bench, and culture in a 37°C incubator;

[0033] Step 2: Prepare seed solution: Prepare seed culture medium, take out the activated culture medium plate after 12 hours of culture, transfer it to the seed culture medium, put it in a 37°C constant temperature shaker, and culture it at a speed of 120 r / min until the OD 600 =0.6;

[0034] Step 3: Determination of colony count: Using the viable cell counting method, take the seed solution and make a 10-fold serial dilution. Spread the final dilution on the activated culture medium and place it in a 37°C incubator for 12 hours. Calculate the number of viable bacteria. The calculated number of viable bacteria is 8×10 7 CFU / mL;

[0035] Step 4: Inoculation of bacteria:

[0036] S41, mixing 10 g of Zanthoxylum bungeanum leaf powder and 15 mL of distilled water, sealing with a sterilization sealing film, sterilizing in a high pressure steam sterilizer at 121° C. for 20 min, taking out after cooling, obtaining a fermentation medium, and storing at room temperature;

[0037] S42, take 15mL seed solution (OD 600 =0.6), operated in a clean bench, centrifuged at 2000rpm for 5min, collected the cells, diluted with 5mL seed culture medium and inoculated into the fermentation medium, stirred and mixed with a flame-sterilized and cooled glass rod, sealed with a sterilized sealing film and placed in a 37°C incubator for fermentation for 3d; the number of viable bacteria in the seed liquid was 8×10 7 CFU / mL, the calculated inoculum size is 1.2×10 8CFU / g (compared to the mass of Zanthoxylum leaf powder), the solid-liquid ratio (mass of Zanthoxylum leaf powder: total volume of distilled water and seed culture medium) was 1 g: 2 mL;

[0038] Step 5, sample collection: After fermentation, take the fermented pepper leaves out of the incubator, put them into a 65°C oven and dry them for about 24 hours until the pepper leaves are powdery when lightly twisted by hand. Collect and seal them in a sample bag and store them at room temperature away from light.

[0039] Example 2

[0040] In this embodiment, in S42, the mixture is sealed with a sterile sealing film and placed in an incubator at 33° C. for fermentation, and the remaining steps are the same as those in Example 1.

[0041] Example 3

[0042] In this embodiment, in S42, the fermentation time is 5 days, and the remaining steps are the same as in Example 1.

[0043] Example 4

[0044] In this embodiment, in S41, the amount of distilled water used is 20 mL, that is, the material-liquid ratio (mass of Zanthoxylum bungeanum powder: total volume of distilled water and seed culture medium) is 1 g:2.5 mL, and the remaining steps are the same as in Example 1.

[0045] Comparative Example 1

[0046] In this comparative example, in S42, the mixture is sealed with a sterile sealing film and placed in an incubator at 39° C. for fermentation, and the remaining steps are the same as those in Example 1.

[0047] Comparative Example 2

[0048] In this comparative example, in S42, the fermentation time is 2 days, and the remaining steps are the same as those in Example 1.

[0049] Comparative Example 3

[0050] In this comparative example, in S41, the amount of distilled water used is 5 mL, that is, the material-liquid ratio (mass of Zanthoxylum bungeanum leaf powder: total volume of distilled water and seed culture medium) is 1 g:1.0 mL, and the remaining steps are the same as in Example 1.

[0051] Example 5

[0052] In this embodiment, S41 is replaced by: take 1000mL of distilled water, add 5g of sucrose, 5g of sorbitol, 0.1g of cyclohexanehexol, 1g of sodium acetate, 0.01g of ZnSO4 and 3g of diammonium hydrogen citrate, stir evenly, adjust the pH to 7.2, and put it into a high-pressure homogenizer for secondary homogenization, that is, first homogenize at 15°C and 30MPa for 5min, and then homogenize at 80°C and 100MPa for 5min to obtain a mixed solution; mix 10g of Zanthoxylum leaf powder and 15mL of the mixed solution, seal with a sterilization sealing film, sterilize at 121°C in a high-pressure steam sterilizer for 20min, take out after cooling, obtain fermentation medium, and store at room temperature; the remaining steps are the same as in Example 1.

[0053] In this embodiment, the composition ratio of the fermentation medium is optimized, which is beneficial to the growth and metabolism of bacteria and promotes the secretion and synthesis of related enzymes such as tannase, thereby improving the ability to degrade tannins; at the same time, a high-pressure homogenizer is used to perform secondary homogenization treatment on the solution, and treatment at different temperatures and pressures is beneficial to improving the uniformity and solubility of each substance in the mixed solution, so that the bacterial cells have more sufficient contact with each substance, thereby further improving the ability to degrade tannins.

[0054] Example 6

[0055] In this embodiment, S41 is replaced by: take 1000mL of distilled water, add 5g of sucrose, 5g of sorbitol, 0.1g of cyclohexanehexol, 1g of sodium acetate, 0.01g of ZnSO4 and 3g of diammonium hydrogen citrate, stir evenly, adjust the pH to 7.2, and obtain a mixed solution; mix 10g of Zanthoxylum bungeanum powder and 15mL of the mixed solution, seal with a sterilization sealing film, sterilize in a high-pressure steam sterilizer at 121°C for 20min, take out after cooling, obtain a fermentation medium, and store at room temperature; the remaining steps are the same as in Example 1.

[0056] Determination of Tannin in Zanthoxylum bungeanum Leaves

[0057] 1. Reagent configuration

[0058] (1) Tannic acid solution: concentration is 50 mg / L. Weigh 50 mg of tannic acid standard (C 76 H 52 O 46 ) is dissolved in 1L of distilled water and put into a reagent bottle for immediate use;

[0059] (2) Acetone solution: acetone: distilled water = 1:1. Take equal volumes of acetone (C3H6O) and distilled water, mix and shake well.

[0060] (3) Sodium carbonate solution: The concentration is 75 g / L. Weigh 37.5 g of anhydrous sodium carbonate (Na2CO3) and dissolve it in 250 mL of warm water. Mix and shake well. After cooling, dilute to 500 mL. Filter into a reagent bottle and store at room temperature for 7 days.

[0061] (4) Sodium tungstate-phosphomolybdic acid mixed solution: weigh 100.0 g sodium tungstate (Na2O4W·2H2O), 20 g phosphomolybdic acid (H3Mo 12 O 40 P·XH2O), dissolved in 750mL distilled water, poured into a 1000mL reflux flask, added 50mL phosphoric acid, mixed and shaken, connected to a condenser reflux tube, heated and refluxed in a boiling water bath for 2h, poured into a 1000mL volumetric flask after cooling, made up to 1000mL with distilled water, shaken and filtered, placed in a brown reagent bottle and stored away from light. It can be stored at room temperature for 14d.

[0062] 2. Sample preparation

[0063] Weigh 0.2 g of sample (accurate to 0.0001 g), place it in a 10 mL centrifuge tube, accurately add 10 mL of acetone solution, seal it, and place it in an ultrasonic extractor at 50 °C for 40 min. After standing, filter it with medium-speed quantitative filter paper, discard the initial filtrate, and collect the subsequent filtrate for measurement. You can also centrifuge it at 3000 rpm for 5 min and collect the supernatant for measurement.

[0064] 3. Drawing of standard curve

[0065] Accurately measure 0.00mL, 0.50mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL and 6.00mL of tannic acid solution, add them into 50mL volumetric flasks containing 30mL of distilled water respectively, and mix and shake well; add 2.5mL of sodium tungstate-phosphomolybdic acid mixed solution, mix and shake well; add 5.0mL of sodium carbonate solution, mix and shake well; then make up to 50mL with water, and mix and shake well. The concentrations of tannic acid standard solutions are: 0.00 mg / L, 0.50 mg / L, 1.00 mg / L, 2.00 mg / L, 3.00 mg / L, 4.00 mg / L, 5.00 mg / L and 6.00 mg / L. After the color development reaction is allowed to proceed at room temperature for 30 min, the absorbance of the standard solution is measured at a wavelength of 760 nm with 0.00 mg / L of the standard curve as blank. A standard curve is plotted with tannic acid concentration as the abscissa and absorbance as the ordinate to obtain the standard curve equation.

[0066] 4. Determination of tannin standard

[0067] The absorbance values ​​of tannin standard solutions with concentrations of 0.00 mg / L, 0.50 mg / L, 1.00 mg / L, 2.00 mg / L, 3.00 mg / L, 4.00 mg / L, 5.00 mg / L and 6.00 mg / L at a wavelength of 760 nm were measured, and the results are shown in Table 1. Tannin standard curve Figure 1 As shown, the calibration curve equation is OD value = 0.0524 × tannin concentration (mg / L) - 0.0032, R 2 is 0.9993.

[0068] Table 1

[0069] Tannin standard solution concentration (mg / L) OD value 0.00 0.0000 0.50 0.0196 1.00 0.0498 2.00 0.0998 3.00 0.1535 4.00 0.2107 5.00 0.2549 6.00 0.3121

[0070] 5. Determination of samples

[0071] Accurately measure 0.1 mL of the sample in a 10 mL centrifuge tube, use a pipette to add 0.25 mL of sodium tungstate-phosphomolybdic acid mixed solution, 0.5 mL of sodium carbonate solution, and 4.15 mL of distilled water, mix and shake well, leave at room temperature for color development for 30 minutes, and use the standard curve 0.00 mg / L as blank at a wavelength of 760 nm to measure the absorbance of the sample solution. Calculate the concentration of tannic acid in the sample solution based on the standard curve. If the absorbance exceeds the range of the standard curve, dilute the sample and repeat the measurement.

[0072] 6. Data processing

[0073] After each experiment, the tannin determination was completed, the measurement data were recorded, and the data were preliminarily sorted and processed through WPS Excel and processed using IBM SPSS 26 software.

[0074] Tannin degradation rate = (tannin content of standard sample - tannin content of sample) / tannin content of standard sample × 100%; among them, the tannin content of Zanthoxylum bungeanum leaf standard sample is 47.288 mg / g.

[0075] The tannin content of the fermented Zanthoxylum bungeanum leaf samples obtained in Examples 1 to 4 and Comparative Examples 1 to 3 was measured, and the results are shown in Table 2. It can be seen that under different fermentation temperatures, fermentation times, and solid-liquid ratios, the tannin degradation rates of Zanthoxylum bungeanum leaves by the methods of Examples 1 to 4 of the present invention are higher than those of Comparative Examples 1 to 3.

[0076] Table 2

[0077] Tannin content (mg / g) Degradation rate (%) Example 1 4.491 90.5 Example 2 5.640 88.1 Example 3 6.523 86.2 Example 4 7.709 83.7 Comparative Example 1 15.355 67.5 Comparative Example 2 10.591 77.6 Comparative Example 3 21.092 55.4

[0078] The tannin content of the fermented Zanthoxylum bungeanum leaf samples obtained in Examples 1, 5-6 was measured, and the results are shown in Table 3. It can be seen that the tannin content of the Zanthoxylum bungeanum leaves was significantly reduced, with a degradation rate of >90%, among which Example 5 had the highest degradation rate, reaching 95.2%.

[0079] Table 3

[0080] Tannin content (mg / g) Degradation rate (%) Example 1 4.491 90.5 Example 5 2.270 95.2 Example 6 3.065 93.5

[0081] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A Bacillus subtilis ( Bacillus subtilis ) strain w-4, the preservation number of the Bacillus subtilis strain w-4 is CCTCC NO: M 2024804.

2. Use of the Bacillus subtilis strain W-4 as claimed in claim 1 in degrading tannins in Zanthoxylum bungeanum leaves.

3. The use according to claim 2, characterized in that The following steps are involved: Step 1: Activation of strains: Prepare activation culture medium plates, take out the stored Bacillus subtilis strain W-4 and thaw it at room temperature, streak the plates in a clean bench, and place them in an incubator for culture; Step 2: Prepare seed solution: prepare seed culture medium, take out the activated culture medium plate after culturing for 10-14 hours, transfer it to the seed culture medium, and culture it in a constant temperature shaker; Step 3: Determination of colony count: Using the viable cell counting method, take the seed solution and perform 10-fold serial dilutions, spread the final dilution on the activated culture medium, place it in an incubator for culture, and calculate the number of viable bacteria; Step 4: Inoculation of bacteria, the method is as follows: S41, mixing Zanthoxylum bungeanum leaf powder and distilled water, sterilizing at 121° C. for 20 min, taking out after cooling, obtaining a fermentation medium, and storing at room temperature; S42. Operate in a clean bench, take the seed solution and centrifuge it at 1500-2500 rpm for 4-7 minutes, collect the bacteria, dilute it with the seed culture medium, and inoculate it into the fermentation medium, stir and mix, and then seal it and put it into the incubator for fermentation; Step 5. Collect samples: After fermentation, take out the fermented pepper leaves, dry them, collect and seal them, and store them at room temperature away from light.

4. The use according to claim 3, characterized in that In the step 1, the Bacillus subtilis strain W-4 is stored at -80°C; the incubator temperature is 33-40°C.

5. The use according to claim 3, characterized in that The specific components and dosage of the activation culture medium are: 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl solid and 20 g / L agar, sterilized at 121° C. for 20 min.

6. The use according to claim 3, characterized in that In step 2, the temperature of the constant temperature shaker is 33-40°C, the speed is 100-150 r / min, and the culture is continued until OD 600 =0.5~0.

7.

7. The use according to claim 3, characterized in that The specific components and dosage of the seed culture medium are: 3 g / L beef extract, 10 g / L peptone and 5 g / L NaCl solid, dissolved in 1000 mL of distilled water, and sterilized at 121° C. for 20 min.

8. The use according to claim 3, characterized in that In the step 3, the culture is carried out by placing the culture medium in an incubator at 33-40° C. for 10-14 hours.

9. The use according to claim 3, characterized in that In the step 4, the mass volume ratio of Zanthoxylum bungeanum leaf powder and distilled water is 1 g: 1.0-2.0 mL; the mixture is sealed and placed in an incubator at 33-37° C. for fermentation for 3-5 days; the inoculation amount of the strain is 8×10 7 ~2×10 8 CFU / g; mass of Zanthoxylum bungeanum leaf powder: total volume of distilled water and seed culture medium is 1g: 1.5~2.5mL.

10. The use according to claim 3, characterized in that In the step 5, the drying is carried out by placing the product in an oven at 60 to 70° C. for 20 to 28 hours.

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