Microbial agent as well as preparation method and application thereof

Abstract

The invention discloses a strain of Enterobacter cancerogenus, which is named as Enterobacter cancerogenus SL12, is preserved in the China Center for Type Culture Collection (CCTCC) on March 18, 2022, and has the preservation number of CCTCC NO: M 2022288. The invention further discloses a microbial agent containing the enterobacter cancerogenus, a preparation method of the microbial agent and application of the microbial agent in the aspect of increasing the content of capsaicin in pepper fruits. The microbial agent containing the enterobacter cancerogenic, the fermentation broth product or the metabolite can significantly improve the capsaicin content in capsicum fruits by 61.9% compared with CK, and has a wide application value in the technical field of improving the capsaicin content in capsicum fruits by applying the microbial agent.

Description

Technical Field

[0001] The present invention belongs to the technical field of green agricultural production, and in particular relates to a microbial agent and a preparation method and application thereof. Background Art

[0002] Capsaicin, found in chili peppers, is an important physiologically active substance, known as "soft gold." It not only imparts peppers with their pungent flavor and is used as a food flavoring and additive, but also possesses antioxidant, anti-inflammatory, antibacterial, antiviral, and immune-modulating properties. It plays a vital role in medicine, military industry, shipbuilding, and agriculture. The current global market demand for capsaicinoids is 60,000 to 70,000 tons annually, while the global production capacity is less than 5,000 tons, resulting in a gap of over 90% to meet demand. Effective solutions are urgently needed to address this issue.

[0003] At present, research on measures to increase the capsaicin content in pepper fruits is mostly focused on cultivating new varieties and improving the environmental conditions for pepper cultivation. Studies have found that increasing the capsaicin content in pepper fruits by applying microbial agents also has broad research value, but there are very few reports on related strains and technical methods. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a cancer-causing Enterobacterium SL12 strain, which has a promoting effect on increasing the capsaicin content of pepper fruits.

[0005] The second object of the present invention is to provide a microbial agent for increasing the capsaicin content in pepper fruits and a preparation method thereof.

[0006] The third object of the present invention is to provide the application of the microbial agent in increasing the capsaicin content in pepper fruits.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] The present invention first provides a cancer-causing Enterobacter SL12, which was deposited in the China Center for Type Culture Collection on March 18, 2022, with a deposit number of CCTCC NO: M 2022288.

[0009] The cancer-causing Enterobacter SL12, the fermentation liquid of the cancer-causing Enterobacter SL12 or the metabolites of the cancer-causing Enterobacter SL12 can increase the capsaicin content in pepper fruits.

[0010] The present invention provides a microbial agent containing the cancer-causing Enterobacter SL12, wherein the microbial agent comprises cancer-causing Enterobacter SL12, a fermentation liquid of cancer-causing Enterobacter SL12, or a metabolite of cancer-causing Enterobacter SL12.

[0011] In some specific embodiments, the fermentation broth of Enterobacter SL12 is an unconcentrated original fermentation broth of Enterobacter SL12, and the effective viable count of the unconcentrated original fermentation broth of Enterobacter SL12 is 6 to 10 billion CFU / mL.

[0012] The present invention also provides a method for preparing the microbial agent, comprising the following steps:

[0013] S1. The cancer-producing Enterobacter SL12 was inoculated into LB solid medium for activation to obtain activated cancer-producing Enterobacter SL12;

[0014] S2. The activated Enterobacter SL12 was inoculated into LB liquid medium for primary fermentation to obtain a primary seed solution of Enterobacter SL12;

[0015] S3. The primary seed solution of Enterobacter SL12 was inoculated into the culture medium for secondary fermentation to obtain a secondary seed solution of Enterobacter SL12;

[0016] S4. Inoculate the secondary seed liquid of Enterobacter carcinogenes SL12 into the culture medium for tertiary fermentation to obtain the microbial agent.

[0017] Furthermore, in step S1, the formula of the LB solid culture medium is: 8-12 g / L peptone, 4-6 g / L yeast extract, 9-11 g / L sodium chloride, 14-16 g / L agar powder, and pH 5.5-8.5.

[0018] The process conditions for activating the cancer-causing Enterobacter SL12 are: temperature 28-35° C., time 1-3 days.

[0019] Furthermore, in step S2, the formula of the LB liquid culture medium is: peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 9-11 g / L, pH 5.5-8.5;

[0020] The primary fermentation process conditions are as follows: liquid volume of 40% to 60%, inoculation amount of 1% to 3%, culture temperature of 28 to 35° C., and shaking culture at 180 to 200 rpm for 16 to 20 hours.

[0021] Furthermore, in step S3 and step S4, the culture medium used in the secondary fermentation is the same as that used in the tertiary fermentation, with a specific formula of: 18-30 g / L glucose, 12-16 g / L potato juice, 20-23 g / L sucrose, 16-22 g / L yeast powder, 2-5 g / L magnesium sulfate, 3-5 g / L disodium hydrogen phosphate, 2-5 g / L sodium chloride, and a pH of 5.5-8.5.

[0022] Furthermore, in step S3, the process conditions of the secondary fermentation are: liquid volume of 50% to 70%, inoculation amount of 4% to 7%, culture temperature of 28 to 35° C., and shaking culture at 180 to 200 rpm for 16 to 20 hours.

[0023] Furthermore, in step S4, the process conditions of the tertiary fermentation are: liquid volume of 50% to 70%, inoculation amount of 4% to 7%, culture temperature of 28 to 35° C., and shaking culture at 180 to 200 rpm for 36 to 48 hours.

[0024] Furthermore, the volume ratio of the primary fermentation, secondary fermentation, and tertiary fermentation fermentation tanks is 1:20:200.

[0025] The present invention also provides a use of the cancer-causing Enterobacter SL12 or the microbial agent as described above or the microbial agent prepared by the preparation method as described above in increasing the capsaicin content in pepper fruits.

[0026] The microbial agent disclosed in the present invention can significantly increase the capsaicin content in pepper fruits, which can be increased by 61.9% compared with the blank control group, thus finding a new direction for effectively increasing the capsaicin content. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the colony morphology of the front side of the plate of Enterobacter cancerogenus SL12;

[0028] Figure 2 This is a scanning electron microscope image of the microscopic morphology of Enterobacter cancerogenus SL12;

[0029] Figure 3 This is the phylogenetic tree analysis of the 16S rDNA sequence of Enterobacter cancerogenus strain SL12;

[0030] Figure 4 The effect of Enterobacter cancerogenus SL12 on the capsaicin content in pepper fruits. DETAILED DESCRIPTION

[0031] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0032] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0033] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0034] Example 1 Isolation and screening of bacteria

[0035] The method for isolating and screening Enterobacter cancerogenus SL12 of the present invention comprises the following steps: collecting fresh, asymptomatic pepper fruits, rinsing them with tap water, and then disinfecting them by soaking them in 5% sodium hypochlorite for 1 minute and 75% alcohol for 30 seconds. After rinsing them with sterile water 2-3 times, the peppers are placed in a sterile mortar and ground with a small amount of sterile water. Endophytic bacteria in the pepper fruits are isolated by a dilution plate method, 100 μL of the dilution solution is evenly spread on LB culture medium, and the mixture is cultured and purified in a 30°C constant temperature incubator. Finally, the strain is preserved in a 25% glycerol suspension at -80°C to obtain a strain of Enterobacter cancerogenus SL12. Figure 1 This is the colony morphology on the front of the plate of Enterobacter cancerogenus SL12.

[0036] Example 2 Classification and Identification of Bacteria

[0037] According to the Manual of Identification of Common Bacteria, the morphological characteristics and physiological and biochemical characteristics of strain SL12 were identified. Strain SL12 grew well on LB medium. The colony morphology was round, with neat edges, smooth surface, translucent, small protrusions, and yellow color. The bacteria were long rod-shaped, such as Figure 2 Physiological and biochemical characteristics of strain SL12: Gram staining is negative, urease is negative, amylase is positive, and it can utilize mannose, glucose, sucrose, and citrate.

[0038] The results of 16S rRNA gene sequencing of the strain are shown in SEQ ID NO.1.

[0039] The strain SL12 was identified using 16S rDNA technology. The 16S rRNA gene was amplified using universal bacterial primers (sequences shown in SEQ ID: 2 and SEQ ID: 3). After PCR amplification, the PCR products were electrophoresed on a 1% agarose gel. The gene products obtained by PCR were sequenced, and the resulting sequences were entered into the NCBI database and compared using BLAST software. Strains with high homology to the strain were selected, and multiple sequence alignment was performed using MEGA 7.0 software to construct a phylogenetic tree. Figure 3 As shown. The 16S rRNA gene sequence of strain SL12 was obtained by PCR amplification. The comparison results showed that the similarity between strain SL12 and Enterobacter sp. reached 98.97%, and its morphological characteristics and physiological and biochemical properties were closest to cancerogens. Therefore, strain SL12 was identified as cancerogens. The strain was named Enterobacter cancerogenus SL12 and was deposited in the China Center for Type Culture Collection, Wuhan (Wuhan University). The strain deposit number is CCTCC NO: M 2022288, and the deposit time is March 18, 2022.

[0040] SEQ ID NO.1:

[0041] CCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGATGAGGTTATTAACCTCACCGCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCGAGAGCAAGCTCTCTGTGCTACC

[0042] SEQ ID NO.2: 27F 5’-AGAGTTTGATCCTGGCTCAG-3’

[0043] SEQ ID NO.3: 1492R 5’-TACGGCTACCTTGTTACGACTT-3’

[0044] Example 3 Preparation of Microbial Inoculum

[0045] This example will introduce the preparation of the microbial inoculum, and the specific process is as follows:

[0046] Strain activation: Inoculate Enterobacter carcinogenes SL12 onto LB solid medium and culture at 28°C for 2 days to obtain activated Enterobacter carcinogenes. The LB solid medium formulation is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, pH 7.0.

[0047] S2. Primary Fermentation (50 mL): Fill the LB medium at 50% capacity and inoculate at 1% inoculum. Inoculate the activated Enterobacter carcinogenes SL12 into LB liquid medium for primary fermentation. Incubate at 28°C with shaking at 180 rpm for 16 hours. This yields the primary seed solution of Enterobacter carcinogenes. The LB liquid medium formulation is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0048] S3. Secondary Fermentation (20 L): A 70% liquid volume was used to inoculate the primary seed solution of Enterobacter carcinogenes into a seed tank for secondary fermentation. The inoculum size was 5%, and the culture temperature was 33°C with shaking at 200 rpm for 16 hours. This yielded the secondary seed solution of Enterobacter carcinogenes. The culture medium used for the secondary fermentation was as follows: 25 g / L glucose, 14 g / L potato juice, 21 g / L sucrose, 18 g / L yeast extract, 3 g / L magnesium sulfate, 4 g / L disodium hydrogen phosphate, and 4 g / L sodium chloride; the pH was 7.

[0049] S4. Tertiary Fermentation (200 L): The fermenter was inoculated with the secondary seed liquid of Enterobacter carcinogenes into a 70% liquid tank for tertiary fermentation at an inoculum size of 5%. The culture temperature was 33°C, with shaking at 200 rpm for 48 hours. The culture medium used for the tertiary fermentation was as follows: 25 g / L glucose, 14 g / L potato juice, 21 g / L sucrose, 18 g / L yeast extract, 3 g / L magnesium sulfate, 4 g / L sodium hydrogen phosphate, and 4 g / L sodium chloride, with a pH of 7. The effective viable count of Enterobacter carcinogenes SL12 obtained by fermentation was 8 billion CFU / mL. After completion of the fermentation in the production tank, the culture medium was directly packaged into a liquid dosage form, which is the microbial inoculum containing Enterobacter carcinogenes SL12.

[0050] Example 4 Application of microbial agents

[0051] XY18 pepper seeds were selected for germination and seedling cultivation. The experiment set up two treatments: microbial agent and CK (clear water). The concentration of the microbial agent containing carcinogenic Enterobacter SL12 was adjusted to 1.0×10 8 CFU / mL.

[0052] XY18 pepper seed seedlings were transplanted at the eight-leaf stage. An equal amount of sterilized substrate nutrient soil was placed in a flowerpot (15 cm × 15 cm). After watering with 20 mL of microbial agent, the seedlings were transplanted into the flowerpot and irrigated with 10 mL of SL12 agent as rooting water.

[0053] Five days after seedling hardening, peppers were simultaneously sprayed and irrigated with a microbial agent at a rate of 20 mL, repeated three times. During the flowering phase, the peppers were sprayed every two days at a rate of 10 mL. The CK group used water instead of the microbial agent, with the same application schedule and dosage as the microbial agent treatment group. Daily water and fertilizer management remained the same for all potted plants.

[0054] Mature green fruits were uniformly collected during the peak fruiting period. Each treatment was repeated three times, and five peppers were selected for each repetition. The pedicles and seeds of the pepper fruits were removed. After drying at 50°C, the capsaicin content in the pepper fruits was determined using GB / T 21266-2007. The results are shown in Table 1.

[0055] Table 1 Effects of microbial agents on capsaicin content in XY18 pepper fruits

[0056]

[0057] Table 1 shows the effect of carcinogenic Enterobacter SL12 microbial agent treatment on the capsaicin content in XY18 pepper fruits. According to the table analysis, the average capsaicin content of XY18 fruits in the microbial agent treatment group is 130.3 mg / g, while the average capsaicin content of XY18 fruits treated with pure water is 80.5 mg / g. It can be seen that the carcinogenic Enterobacter SL12 microbial agent can significantly increase the capsaicin content in pepper fruits, which is 61.9% higher than CK (pure water). Figure 4 This is a bar chart showing the capsaicin content in pepper fruits treated with the carcinogenic Enterobacter SL12 microbial agent and compared with the water control. In the figure, "*" indicates a significant difference between the treatments (P<0.05).

Claims

1. A strain of Enterobacter carcinogenes SL12, which was deposited in the China Center for Type Culture Collection on March 18, 2022, with the deposit number CCTCC NO: M 2022288, characterized in that: The cancer-causing Enterobacter SL12, the fermentation liquid of the cancer-causing Enterobacter SL12 or the metabolites of the cancer-causing Enterobacter SL12 can increase the capsaicin content in pepper fruits.

2. A microbial agent comprising the carcinogenic Enterobacter SL12 according to claim 1, characterized in that: The microbial agent includes cancer-causing Enterobacter SL12, fermentation liquid of cancer-causing Enterobacter SL12 or metabolites of cancer-causing Enterobacter SL12.

3. The microbial agent according to claim 2, characterized in that The fermentation liquid of the cancer-causing Enterobacter SL12 is an unconcentrated original fermentation liquid of the cancer-causing Enterobacter SL12, and the effective viable bacteria count of the unconcentrated original fermentation liquid of the cancer-causing Enterobacter SL12 is 6 to 10 billion CFU / mL.

4. A method for preparing the microbial agent according to any one of claims 2 or 3, characterized in that: The following steps are involved: S1. The cancer-producing Enterobacter SL12 was inoculated into LB solid medium for activation to obtain activated cancer-producing Enterobacter SL12; S2. The activated Enterobacter SL12 was inoculated into LB liquid medium for primary fermentation to obtain a primary seed solution of Enterobacter SL12; S3. The primary seed solution of Enterobacter SL12 was inoculated into the culture medium for secondary fermentation to obtain a secondary seed solution of Enterobacter SL12; S4. Inoculate the secondary seed liquid of Enterobacter carcinogenes SL12 into the culture medium for tertiary fermentation to obtain the microbial agent.

5. The method for preparing the microbial agent according to claim 4, characterized in that: In step S1, the formula of the LB solid culture medium is: 8-12 g / L peptone, 4-6 g / L yeast extract, 9-11 g / L sodium chloride, 14-16 g / L agar powder, and pH 5.5-8.5; The process conditions for activating the cancer-causing Enterobacter SL12 are: temperature 28-35° C., time 1-3 days.

6. The method for preparing the microbial agent according to claim 4, characterized in that: In step S2, the formula of the LB liquid culture medium is: 8-12 g / L peptone, 4-6 g / L yeast extract, 9-11 g / L sodium chloride, and pH 5.5-8.5; The primary fermentation process conditions are as follows: liquid volume of 40% to 60%, inoculation amount of 1% to 3%, culture temperature of 28 to 35° C., and shaking culture at 180 to 200 rpm for 16 to 20 hours.

7. The method for preparing the microbial agent according to claim 4, characterized in that: In step S3 and step S4, the culture medium used in the secondary fermentation is the same as that used in the tertiary fermentation, with a specific formula of: 18-30 g / L of glucose, 12-16 g / L of potato juice, 20-23 g / L of sucrose, 16-22 g / L of yeast powder, 2-5 g / L of magnesium sulfate, 3-5 g / L of disodium hydrogen phosphate, 2-5 g / L of sodium chloride, and a pH of 5.5-8.

5.

8. The method for preparing the microbial agent according to claim 4, characterized in that: In step S3, the process conditions of the secondary fermentation are: liquid volume of 50% to 70%, inoculation amount of 4% to 7%, culture temperature of 28 to 35° C., shaking culture at 180 to 200 rpm for 16 to 20 hours; In step S4, the process conditions of the tertiary fermentation are: liquid volume of 50% to 70%, inoculation amount of 4% to 7%, culture temperature of 28 to 35° C., and shaking culture at 180 to 200 rpm for 36 to 48 hours.

9. The method for preparing the microbial agent according to claim 4, characterized in that: In step S3, the volume ratio of the primary fermentation, secondary fermentation, and tertiary fermentation fermentation tanks is 1:20:

200. Use of the cancer-causing Enterobacter SL12 according to claim 1, the microbial agent according to any one of claims 2 to 3, or the microbial agent prepared by the preparation method according to any one of claims 4 to 9 in increasing the capsaicin content in pepper fruits.

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