Bacillus subtilis and application thereof in flue-cured tobacco planting

Abstract

The invention discloses bacillus subtilis and application thereof in flue-cured tobacco planting, and belongs to the technical field of microorganisms. The preservation number of the bacillus subtilis GUSC13 provided by the invention is CCTCC (China Center for Type Culture Collection) NO: M 2025063. The bacillus subtilis GUSC13 can improve the drought resistance of the flue-cured tobacco. Experimental studies show that after the flue-cured tobacco is irrigated with a bacterial solution of the bacillus subtilis GUSC13 and is subjected to drought treatment, compared with a control group, the oxidation resistance of the flue-cured tobacco and the content of osmotic regulation substances can be remarkably improved, the content of malondialdehyde is reduced, the root activity is improved, and the root growth is promoted; in the rehydration process, the tobacco can be promoted to recover normal physiological functions more quickly. Besides, after the bacterial liquid of the bacillus subtilis GUSC13 is irrigated to the stem bases of the flue-cured tobaccos, the content of components such as total sugar, nicotine and total nitrogen of the flue-cured tobaccos after the flue-cured tobaccos are harvested can be obviously changed, and the quality of flue-cured tobacco products is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly to a Bacillus subtilis and its application in flue-cured tobacco cultivation. Background Art

[0002] In recent years, with global warming, natural disasters such as high temperature, drought and flood have occurred frequently, seriously affecting crop production. Among them, drought has the most serious impact on the growth, yield and quality of crops, making drought one of the main climate disasters threatening agricultural production in China and the world. Flue-cured tobacco is an important economic crop in China and plays an important role in the economic development of the country and regions. Drought stress will lead to a decrease in the yield of flue-cured tobacco, an increase in the total nitrogen and nicotine content of tobacco leaves, and a decrease in the reducing sugar content, seriously affecting the production of high-quality flue-cured tobacco in China.

[0003] Potassium-solubilizing bacteria are a type of bacteria that can decompose potassium-containing minerals. They can decompose and convert insoluble mineral potassium in the soil into available potassium for direct absorption and utilization by plants. At the same time, they also have the functions of increasing crop yield, improving crop quality, improving soil fertility and enhancing crop stress resistance. However, there are few reports on the improvement of crop drought resistance by potassium-solubilizing bacteria at present. Therefore, studying the effect of potassium-solubilizing bacteria on crop drought resistance is of great significance for crop production. Summary of the Invention

[0004] The purpose of the present invention is to provide a Bacillus subtilis and its application in flue-cured tobacco cultivation to solve the problems existing in the above-mentioned prior art. After irrigating the bacterial liquid containing the Bacillus subtilis provided by the present invention, the drought resistance of flue-cured tobacco can be significantly improved, and the quality of flue-cured tobacco products can also be improved.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a Bacillus subtilis GUSC13, which has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2025063, the deposit date being January 8, 2025, and the deposit address being Wuhan University, Wuhan, China.

[0007] The present invention provides a microbial inoculant containing the Bacillus subtilis GUSC13.

[0008] The present invention also provides an application of the Bacillus subtilis GUSC13 or the microbial inoculant in improving plant drought resistance.

[0009] Preferably, the plant is flue-cured tobacco.

[0010] The present invention also provides a method for improving the drought resistance of plants, which includes the step of irrigating the roots of the plants with the bacterial solution of the Bacillus subtilis GUSC13 described above.

[0011] Preferably, the dosage of the bacterial solution is not less than 60 mL; the viable bacteria count in the bacterial solution is not less than 1×10 8 CFU / mL.

[0012] Preferably, the plant is flue-cured tobacco.

[0013] The present invention also provides an application of the Bacillus subtilis GUSC13 described above or the microbial inoculant in improving the quality of flue-cured tobacco products.

[0014] The present invention also provides a method for improving the quality of flue-cured tobacco products, which includes the step of irrigating the base of the stem of flue-cured tobacco with the bacterial solution of the Bacillus subtilis GUSC13 described above.

[0015] Preferably, the dosage of the bacterial solution is 100 - 200 mL; the viable bacteria count in the bacterial solution is not less than 1×10 8 CFU / mL.

[0016] The present invention discloses the following technical effects:

[0017] The Bacillus subtilis GUSC13 provided by the present invention can improve the drought resistance of flue-cured tobacco. Experimental studies have shown that when irrigating the roots of flue-cured tobacco with the bacterial solution of GUSC13, after drought treatment, compared with the control, it can significantly improve the antioxidant capacity and the content of osmoregulatory substances of flue-cured tobacco, reduce the content of malondialdehyde, improve the root activity and promote root growth; during the rehydration process, it can also promote the tobacco to recover normal physiological functions faster. In addition, after irrigating the base of the stem of flue-cured tobacco with the bacterial solution of Bacillus subtilis GUSC13, it can also significantly change the contents of total sugar, salt and total nitrogen in the cured tobacco leaves after baking, and improve the quality of flue-cured tobacco products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 It is a multi-gene phylogenetic tree diagram of strain GUSC13 constructed based on 16S rDNA and gyrA gene sequences;

[0020] Figure 2Statistical chart of the effect of Bacillus subtilis GUSC13 on the root activity of flue-cured tobacco under drought and rehydration treatments; among them, A is the drought treatment; B is the rehydration treatment; different letters indicate that the results are significantly different statistically.

[0021] Figure 3 Statistical chart of the effect of Bacillus subtilis GUSC13 on the antioxidant enzyme activities of flue-cured tobacco under drought and rehydration treatments; among them, A is the superoxide dismutase activity under drought treatment; B is the catalase activity under drought treatment; C is the peroxidase activity under drought treatment; D is the superoxide dismutase activity under rehydration treatment; E is the catalase activity under rehydration treatment; F is the peroxidase activity under rehydration treatment; different letters indicate that the results are significantly different statistically.

[0022] Figure 4 Statistical chart of the effect of Bacillus subtilis GUSC13 on the malondialdehyde content of flue-cured tobacco under drought and rehydration treatments; among them, A is the drought treatment; B is the rehydration treatment; different letters indicate that the results are significantly different statistically.

[0023] Figure 5 Statistical chart of the effect of Bacillus subtilis GUSC13 on the content of osmotic adjustment substances in flue-cured tobacco under drought and rehydration treatments; among them, A is the proline content under drought treatment; B is the soluble sugar content under drought treatment; C is the soluble protein content under drought treatment; D is the potassium content under drought treatment; E is the proline content under rehydration treatment; F is the soluble sugar content under rehydration treatment; G is the soluble protein content under rehydration treatment; H is the potassium content under rehydration treatment; different letters indicate that the results are significantly different statistically.

[0024] Figure 6 Observation diagram of the effect of Bacillus subtilis GUSC13 on the phenotype of flue-cured tobacco after drought treatment. Detailed implementation mode

[0025] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0030] Isolation and Identification of the Strain in Example 1

[0031] 1. Isolation and Purification of the Strain

[0032] Take the rhizosphere soil of grapes and bring it back to the laboratory to isolate the strain. Weigh 10 g of soil sample and add it to 90 mL of sterile water. Mix well, let it stand for 5 min, and dilute it by gradient to 10 -7 , and respectively pipette 0.2 mL of bacterial liquid from 3 dilution gradients (10 -5 , 10 -6 , 10 -7 ) and add them to the selective medium petri dishes. Spread evenly, with 3 replicates for each dilution gradient. Invert the petri dishes and culture them in an incubator at 30 °C for 4 - 5 d. Select colonies with good growth potential that can produce potassium-dissolving circles (i.e., potassium-dissolving bacteria), streak and purify them, with 3 replicates, until pure-cultured potassium-dissolving bacteria are obtained, and store them in a refrigerator at 4 °C.

[0033] 2. Molecular Biology Identification of the Strain

[0034] The DNA of the antagonistic strain was extracted using a bacterial genomic DNA extraction kit (Shanghai Qingke Biotechnology Co., Ltd.). After PCR amplification of the bacterial DNA extracted using the bacterial universal primers 16S rDNA and gyrA, electrophoresis detection was performed using 1.0% (mass fraction) agarose gel. The PCR amplification products were sent to Sangon Biotech (Beijing) Co., Ltd. for sequencing. After the sequencing results were corrected, a preliminary BLAST comparison was performed on NCBI. According to the comparison results, sequences with relatively close genetic relationships and type strains were downloaded. After correcting the sequences of each strain on the Bio Edit 7.0 software, the MEGA 11 software was used and the maximum likelihood method (Maximum Likelihood, ML) was adopted to construct a multi-gene phylogenetic tree.

[0035] Construction of a multi-gene phylogenetic tree of strain GUSC13 based on 16S rDNA and gyrA gene sequences ( Figure 1 ), the results showed that strain GUSC13 clustered with Bacillus subtilis NRRL B-4219, and the support rate was 100%; therefore, strain GUSC13 was identified as Bacillus subtilis.

[0036] 3. Preservation of the strain

[0037] Bacillus subtilis GUSC13 has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2025063, the deposit date being January 8, 2025, and the deposit address being Wuhan University, Wuhan, China.

[0038] Example 2 Determination of the potassium-solubilizing efficiency of the strain

[0039] 1. Cultivation of bacteria

[0040] The mother liquor of activated Bacillus subtilis GUSC13 was inoculated into liquid LB medium and cultured with shaking at 37°C and 200 r / min for 12 h. After centrifugation, the supernatant was discarded, and the bacterial cells were washed twice with sterile water (washing away the medium). The collected bacteria were suspended with sterile water to prepare a bacterial suspension (OD 600 about 0.8). 5 mL of the bacterial suspension was inoculated into a 250 mL Erlenmeyer flask containing 95 mL of potassium-deficient medium (containing 1 g of potassium feldspar powder), and the same volume of sterile water was added as a blank control. The mixture was cultured with shaking at 37°C and 200 r / min for 7 d.

[0041] 2. Measurement of potassium after the medium is treated with H 2 O 2 treatment

[0042] Take 10 mL of the bacterial culture solution and add 2 mL of 6% H 2 O 2 , digest it in a boiling water bath for 1 h, centrifuge the digestion solution at 13000 r / min for 5 min, take the supernatant and measure the K + concentration. The potassium standard solution is prepared with a potassium-deficient medium without potassium feldspar powder.

[0043] 3. Plotting of the potassium standard curve

[0044] For the prepared potassium standard series solutions, use the potassium standard solution containing K + 0 μg / mL to adjust the galvanometer reading on the flame photometer to 0, and then measure them in sequence from dilute to concentrated, and record the galvanometer readings. Take the galvanometer readings as the ordinate and the mass concentration of potassium as the abscissa to plot the standard curve.

[0045] 4. Detection results of potassium-solubilizing efficiency

[0046] Calculate the K + content in the culture solution through the standard curve, and then calculate the potassium-solubilizing efficiency. Table 1 shows that the potassium-solubilizing efficiency of Bacillus subtilis GUSC13 is 158.95%.

[0047] Table 1 Potassium-solubilizing efficiency of Bacillus subtilis GUSC13

[0048]

[0049] Example 3 Pot experiment under drought conditions

[0050] 1. Experimental method

[0051] After 7 days of slow seedling stage after transplantation, use the bacterial solution of Bacillus subtilis GUSC13 (the viable bacteria count is 1×10 8The first root irrigation treatment was carried out at a concentration of [[ID=]], and the second root irrigation treatment was carried out after an interval of 5 days. Each plant was irrigated with 60 mL each time. After the second root irrigation, drought stress was started using the weighing method, and the relative soil water content was controlled at 30%-35%. After sampling on the 15th day of drought treatment, rehydration was carried out, and the relative soil water content of all treatments during rehydration was controlled at 70%-80%. There were 30 pots for each treatment, repeated 3 times, for a total of 360 tobacco seedlings. At 0 d, 2 d, 5 d, 10 d, and 15 d of drought treatment, and 12 h, 36 h, 72 h, and 168 h after rehydration treatment, the leaves at the 2nd and 3rd leaf positions from the top of the tobacco seedlings were mixed for determination of antioxidant enzyme activity, osmotic adjustment substances, and malondialdehyde content. Samples of the root tip parts of the tobacco plants were taken to measure root activity. Each time 6 tobacco seedlings were taken for each treatment during sampling. At the 15th day of drought treatment and the 168th h of rehydration treatment, 3 tobacco seedlings were randomly selected for each treatment, and the leaves at the 2nd and 3rd leaf positions from the top, roots, and soil of the tobacco plants were taken to measure the chloroplast pigment content of the tobacco seedling leaves, root scanning, and soil nutrient content respectively.

[0052] 2. Experimental results

[0053] The observed results of the phenotypes of tobacco seedlings after treatment are as Figure 6 shown.

[0054] The results of root activity are as Figure 2 shown. Compared with CK, the root activity of flue-cured tobacco treated with Bacillus subtilis GUSC13 increased significantly at 0 d, 2 d, 5 d, 10 d, and 15 d of drought, with the increase rates being 53.19%, 23.87%, 50.68%, 12.64%, and 29.42% respectively. It also increased significantly at 12 h, 36 h, 72 h, and 168 h after rehydration, with the increase rates being 7.50%, 11.70%, 15.15%, and 18.00% respectively.

[0055] The results of root scanning are shown in Table 2. At 15 d of drought stress, compared with CK, the total root length, total root surface area, root diameter, total root volume, and number of root tips of the flue-cured tobacco seedlings treated with Bacillus subtilis GUSC13 increased significantly by 9.19%, 37.51%, 29.92%, 77.86%, and 64.08% respectively. At 168 h of rehydration, the total root length, total root surface area, total root volume, and number of root tips of the treatment with Bacillus subtilis GUSC13 were all significantly increased compared with CK, with the increase rates being 28.50%, 20.50%, 13.56%, and 102.10% respectively.

[0056] Table 2 Effects of Bacillus subtilis GUSC13 on the root growth of flue-cured tobacco seedlings under drought stress

[0057]

[0058] Note: Different letters indicate significant differences statistically.

[0059] Table 3 Effects of Bacillus subtilis GUSC13 on the root growth of flue-cured tobacco seedlings after rehydration

[0060]

[0061] Note: Different letters indicate statistically significant differences.

[0062] The results of photosynthetic pigments are shown in Tables 4 and 5. At 15 d of drought stress, the values of chlorophyll a, chlorophyll b, carotenoid, and chlorophyll a / b in the treatment with Bacillus subtilis GUSC13 were significantly increased compared with CK, with the increase rates being 17.52%, 5.97%, 16.93%, 13.97%, and 10.66% respectively. At 168 h of rehydration, the contents of chlorophyll a, chlorophyll a, and total chlorophyll in the treatment with Bacillus subtilis GUSC13 were significantly increased by 8.38%, 6.24%, and 7.86% compared with CK.

[0063] Table 5 Effects of Bacillus subtilis GUSC13 on the photosynthetic pigment contents in the leaves of flue-cured tobacco seedlings under drought stress

[0064]

[0065] Note: Different letters indicate statistically significant differences.

[0066] Table 6 Effects of Bacillus subtilis GUSC13 on the photosynthetic pigment contents in the leaves of flue-cured tobacco seedlings after rehydration

[0067]

[0068] Note: Different letters indicate statistically significant differences.

[0069] The results of antioxidant enzymes are as Figure 3 shown. The activities of superoxide dismutase in the leaves of flue-cured tobacco treated with Bacillus subtilis GUSC13 were significantly increased by 19.26%, 17.59%, 12.34%, and 10.30% compared with CK at 2 d, 5 d, 10 d, and 15 d after drought ( Figure 3 in A); the activities of catalase in the leaves of flue-cured tobacco treated with Bacillus subtilis GUSC13 were significantly increased by 23.68%, 19.65%, 31.31%, 23.52%, and 16.77% compared with CK at 0 d, 2 d, 5 d, 10 d, and 15 d after drought ( Figure 3 in B); the activities of peroxidase in the leaves of flue-cured tobacco treated with Bacillus subtilis GUSC13 were significantly increased by 16.68%, 11.99%, 9.99%, and 10.23% compared with CK at 2 d, 5 d, 10 d, and 15 d after drought ( Figure 3In C). After rehydration, the decline rates of the contents of superoxide dismutase, catalase, and peroxidase in the flue-cured tobacco leaves treated with Bacillus subtilis GUSC13 were all higher than those of the CK ( Figure 3 In D-F).

[0070] The results of the malondialdehyde content showed that the malondialdehyde content in the leaves of flue-cured tobacco seedlings treated with Bacillus subtilis GUSC13 was significantly reduced by 17.97%, 16.53%, and 8.16% compared with the CK at 5 d, 10 d, and 15 d of drought ( Figure 4 In A); at 12 h, 36 h, 72 h, and 168 h after rehydration, it was significantly reduced by 10.81%, 13.15%, 5.38%, and 14.02% compared with the CK ( Figure 4 In B).

[0071] The results of the osmoregulatory substances showed that the proline content in the leaves of flue-cured tobacco seedlings treated with Bacillus subtilis GUSC13 was significantly increased by 63.92%, 39.99%, 12.39%, 18.97%, and 11.16% compared with the CK at 0 d, 2 d, 5 d, 10 d, and 15 d of drought ( Figure 5 In A); the soluble sugar content in the leaves of flue-cured tobacco seedlings treated with Bacillus subtilis GUSC13 was significantly increased by 41.56%, 16.82%, and 62.96% compared with the CK at 5 d, 10 d, and 15 d of drought ( Figure 5 In B); the soluble protein content in the leaves of flue-cured tobacco seedlings treated with Bacillus subtilis GUSC13 was significantly increased by 16.21%, 13.37%, 5.87%, 7.91%, and 9.35% compared with the CK at 0 d, 2 d, 5 d, 10 d, and 15 d of drought ( Figure 5 In C); the potassium content in the leaves of flue-cured tobacco seedlings treated with Bacillus subtilis GUSC13 was significantly increased by 4.32%, 4.92%, 5.84%, and 5.74% compared with the CK at 2 d, 5 d, 10 d, and 15 d of drought ( Figure 5 In D). After rehydration, the contents of proline and soluble sugar in the flue-cured tobacco leaves treated with Bacillus subtilis GUSC13 decreased faster, the soluble protein content decreased first and then increased and was significantly higher than that of the control, and the potassium content increased after rehydration and was always higher than that of the CK ( Figure 5 In E-H).

[0072] Example 4 Field plot experiment

[0073] In this example, water was used as the control, each treatment was repeated 3 times, and there were 60 flue-cured tobacco plants in each plot. The Bacillus subtilis bacterial solution used in this example was prepared from 1 kg of Bacillus subtilis bacterial agent (the bacterial content was 1×10 10It is prepared by diluting with 100 kg of water at (CFU / g); 100 - 200 mL of the bacterial liquid is watered to the base of the tobacco plant stem for each plant (when the soil humidity is greater than 50%, 100 mL of the liquid medicine is applied, and when the soil humidity is less than 50%, 200 mL of the liquid medicine is applied). The first root irrigation is carried out after the tobacco seedlings are transplanted and survive, and the root irrigation is carried out once every 25 days for a total of 3 times. Other field management measures are all implemented according to the local production plan.

[0074] After the flue-cured tobacco is harvested and baked by plot, it is graded (the grading standard follows GB 2635-1992). After grinding the baked tobacco leaf samples of B2F, C3F, and X2F of each treatment, the contents of total sugar, reducing sugar, nicotine, total nitrogen, chlorine, and potassium in the baked tobacco leaves are determined by the method of near-infrared scanning, and the nitrogen-nicotine ratio, sugar-nicotine ratio, two-sugar ratio, and potassium-chlorine ratio are calculated.

[0075] The results show that:

[0076] The detection results of the tobacco leaves of different grades of flue-cured tobacco are shown in Tables 7 and 8.

[0077] The results of the baked tobacco leaves of B2F grade show that the treatment with Bacillus subtilis GUSC13 can significantly increase the contents of total sugar, reducing sugar, potassium, and chlorine in the baked tobacco leaves of B2F grade, as well as the nitrogen-nicotine ratio, sugar-nicotine ratio, two-sugar ratio, and potassium-chlorine ratio.

[0078] The results of the baked tobacco leaves of C3F grade show that the treatment with Bacillus subtilis GUSC13 can significantly increase the contents of total nitrogen, nicotine, and potassium in the baked tobacco leaves of C3F grade, as well as the nitrogen-nicotine ratio, two-sugar ratio, and potassium-chlorine ratio.

[0079] The results of the baked tobacco leaves of X2F grade show that the treatment with Bacillus subtilis GUSC13 can significantly increase the contents of total nitrogen, nicotine, and potassium in the baked tobacco leaves of X2F grade, as well as the two-sugar ratio and potassium-chlorine ratio.

[0080] Table 7 Chemical components of baked tobacco leaves of different grades

[0081]

[0082] Note: Different letters indicate significant differences statistically.

[0083] Table 8 Derived values of chemical components of baked tobacco leaves of different grades

[0084]

[0085] Note: Different letters indicate significant differences statistically.

[0086] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Bacillus subtilis GUSC13, characterized in that: The Bacillus subtilis GUSC13 has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 2025063, a deposit date of January 8, 2025, and a deposit address of Wuhan University, Wuhan, China.

2. A microbial agent, characterized in that: The method comprises the Bacillus subtilis GUSC13 according to claim 1.

3. Use of the Bacillus subtilis GUSC13 as claimed in claim 1 or the microbial agent as claimed in claim 2 in improving the drought resistance of plants.

4. The use according to claim 3, characterized in that The plant is flue-cured tobacco.

5. A method for improving drought resistance of plants, characterized in that: The method comprises the step of using the bacterial solution of Bacillus subtilis GUSC13 described in claim 1 to perform root irrigation treatment on plants.

6. The method according to claim 5, characterized in that The amount of the bacterial solution is not less than 60 mL; the number of viable bacteria in the bacterial solution is not less than 1×10 8 CFU / mL.

7. The method according to claim 5 or 6, characterized in that The plant is flue-cured tobacco.

8. Use of the Bacillus subtilis GUSC13 according to claim 1 or the microbial agent according to claim 2 in improving the quality of flue-cured tobacco products.

9. A method for improving the quality of flue-cured tobacco products, characterized in that: The method comprises the step of using the bacterial liquid of Bacillus subtilis GUSC13 described in claim 1 to water the base of the flue-cured tobacco stem.

10. The method according to claim 9, characterized in that The dosage of the bacterial solution is 100-200 mL; the number of viable bacteria in the bacterial solution is not less than 1×10 8 CFU / mL.

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