Microbial agent, preparation method and application thereof

Abstract

The application discloses a strain of Enterobacter cancerogenus, named Enterobacter cancerogenus SL12, which is preserved in the China Center for Type Culture Collection on March 18, 2022, and has a preservation number of CCTCC NO: M 2022288. The application also discloses a microbial inoculum containing the Enterobacter cancerogenus, a preparation method of the microbial inoculum and application of the microbial inoculum in increasing the content of capsaicin in pepper fruits. The microbial inoculum containing the Enterobacter cancerogenus, fermentation liquid products or metabolic products can significantly increase the content of capsaicin in pepper fruits, and the content of capsaicin is increased by 61.9% compared with CK, so that the application has wide application value in the technical field of increasing the content of capsaicin in pepper fruits by applying the microbial inoculum.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of agricultural green production, and particularly relates to a microbial agent and a preparation method and application thereof. BACKGROUND

[0002] Capsaicin in pepper fruits is an important physiological active substance, which is known as "soft gold". Capsaicin not only gives pepper a pungent taste for use as a food flavoring agent and additive, but also has physiological functions such as antioxidant, anti-inflammatory, antibacterial, antiviral, and immune regulation, and plays an important role in the fields of medicine, military, ship corrosion prevention, and agriculture. At present, the global annual demand for capsaicin substances is 60,000-70,000 tons, while the global total production capacity is less than 5,000 tons, with a demand gap of more than 90%, and effective measures are urgently needed to solve this problem.

[0003] At present, the research on measures to improve the content of capsaicin in pepper fruits mainly focuses on cultivating new varieties and improving the cultivation environment of peppers. It is found that improving the content of capsaicin in pepper fruits by applying microbial agents also has wide research value, but there are few reports on related strains and technical methods. SUMMARY

[0004] One of the purposes of the present application is to provide a strain of Enterobacter cancerogenus SL12, which has a promoting effect on improving the content of capsaicin in pepper fruits.

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

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

[0007] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0008] The present application first provides a strain of Enterobacter cancerogenus SL12, which was preserved in the China Center for Type Culture Collection on March 18, 2022, and the preservation number is CCTCC NO: M 2022288.

[0009] The Enterobacter cancerogenus SL12, the fermentation broth of Enterobacter cancerogenus SL12, or the metabolites of Enterobacter cancerogenus SL12 can improve the content of capsaicin in pepper fruits.

[0010] The present application provides a microbial agent containing the Enterobacter cancerogenus SL12, which comprises the Enterobacter cancerogenus SL12, the fermentation broth of Enterobacter cancerogenusSL12, or the metabolites of Enterobacter cancerogenus SL12.

[0011] In some specific embodiments, the fermentation broth of Enterobacter carcinogens SL12 is the unconcentrated original fermentation broth of Enterobacter carcinogens SL12, and the effective viable count of the unconcentrated original fermentation broth of Enterobacter carcinogens SL12 is 60 to 100 billion CFU / mL.

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

[0013] S1. Enterobacter SL12 carcinogenic bacteria were inoculated into LB solid medium for activation to obtain activated Enterobacter SL12 carcinogenic bacteria;

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

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

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

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

[0018] The activation conditions for the *Enterobacter carcinogens* SL12 are: temperature 28–35°C, time 1–3 days.

[0019] Furthermore, in step S2, the formulation 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.

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

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

[0022] Further, in step S3, the process conditions of the secondary fermentation are as follows: liquid loading amount 50%-70%, inoculation amount 4%-7%, culture temperature 28-35 DEG C, 180-200 rpm shock culture for 16-20h.

[0023] Further, in step S4, the process conditions of the tertiary fermentation are as follows: liquid loading amount 50%-70%, inoculation amount 4%- 7%, culture temperature 28-35 DEG C, 180-200 rpm shock culture 36-48h.

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

[0025] The application also provides application of the Enterobacter cancerogenus SL12 or the microbial inoculant prepared by the preparation method to increasing the content of capsaicin in pepper fruits.

[0026] The microbial inoculant can significantly increase the content of capsaicin in pepper fruits, and can increase the content of capsaicin by 61.9% compared with a blank control group, thereby finding a new direction for effectively increasing the content of capsaicin. BRIEF DESCRIPTION OF DRAWINGS

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

[0028] Figure 2 It is a scanning electron microscope scanning diagram of the micro morphology of the Enterobacter cancerogenus SL12;

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

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

[0031] In order to facilitate understanding of the application, the application will be described more fully and completely by reference to the drawings and preferred embodiments below, but the scope of protection of the application is not limited to the following specific embodiments.

[0032] Unless otherwise defined, 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 application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

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

[0034] Example 1 Isolation and screening of bacteria

[0035] The isolation and screening method of Enterobacter cancerogenus SL12 of the present application is as follows: fresh and healthy pepper fruits are collected, washed with tap water, soaked in 5% sodium hypochlorite for 1 minute, and then soaked in 75% alcohol for 30 seconds for disinfection. After washing with sterile water for 2-3 times, the pepper is placed in a sterile mortar, a small amount of sterile water is added for grinding, and the endophytic bacteria in the pepper fruits are isolated by dilution and spread plate method. 100 μL of the diluent is uniformly spread on LB medium and cultured in a 30°C constant temperature incubator for purification. Finally, the strain of Enterobacter cancerogenus SL12 is preserved in 25% glycerol suspension at -80°C. Figure 1 The plate front colony morphology of Enterobacter cancerogenus SL12.

[0036] Example 2 Classification and identification of bacteria

[0037] According to the Handbook of Systematic Identification of Common Bacteria, the strain SL12 is identified for morphological characteristics and physiological and biochemical properties. The strain SL12 grows well on LB medium, and the colony morphology is round, with neat edges, smooth surface, semi-transparency, small protuberance, and yellow color. The bacterial body is long rod-shaped, as shown in FIG. 1. Figure 2 The physiological and biochemical properties of the strain SL12 are as follows: gram staining is negative, urease is negative, amylase is positive, and the strain can utilize mannose, glucose, sucrose and citrate.

[0038] The 16S rRNA gene sequence determination result of the strain is shown in SEQ ID NO. 1.

[0039] Strain SL12 was identified by 16S rDNA identification technology. The 16S rRNA gene was amplified by using bacterial universal primers (sequences shown in SEQ ID: 2 and SEQ ID: 3). After PCR amplification, the PCR product was detected by 1% agarose gel electrophoresis. The gene product obtained by PCR was sequenced, the obtained sequence was input into NCBI database, and BLAST software was used for comparison and analysis, strains with higher homology with the strain were selected, MEGA 7.0 software was used for multiple sequence alignment and establishment of phylogenetic tree, see Figure 3 The 16S rRNA gene sequence of strain SL12 was obtained by PCR amplification. The alignment results show that the similarity of strain SL12 and Enterobacter sp. reaches 98.97%, and its morphological characteristics and physiological and biochemical characteristics are most similar to Enterobacter cancerogenus. Therefore, strain SL12 is identified as Enterobacter cancerogenus. The strain is named Enterobacter cancerogenus (Enterobacter cancerogenus) SL12, preserved in China Center for Type Culture Collection, Wuhan (Wuhan University), and the strain preservation number is CCTCC NO: M 2022288, and the preservation 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 inoculant

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

[0046] S1. Strain activation: Enterobacter cancerogenus SL12 was inoculated on LB solid medium and incubated at 28°C for 2 days to obtain activated Enterobacter cancerogenus. The formula of LB solid medium is as follows: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 15 g / L of agar, and pH 7.0.

[0047] S2. Primary fermentation (50 mL): the liquid volume was 50%, and the inoculation amount was 1%. The activated Enterobacter cancerogenus SL12 was inoculated in LB liquid medium for primary fermentation, and the culture temperature was 28°C, and the shaking culture was performed at 180 rpm for 16 h. The primary seed liquid of Enterobacter cancerogenus was obtained. The formula of LB liquid medium is as follows: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, and pH 7.0.

[0048] S3. Secondary fermentation (20 L): the liquid volume was 70%, and the primary seed liquid of Enterobacter cancerogenus was inoculated in the seed tank for secondary fermentation, and the inoculation amount was 5%. The culture temperature was 33°C, and the shaking culture was performed at 200 rpm for 16 h. The secondary seed liquid of Enterobacter cancerogenus was obtained. The formula of the medium used in the secondary fermentation is as follows: 25 g / L of glucose, 14 g / L of potato juice, 21 g / L of sucrose, 18 g / L of yeast powder, 3 g / L of magnesium sulfate, 4 g / L of disodium hydrogen phosphate, 4 g / L of sodium chloride, and pH 7.

[0049] S4. Tertiary fermentation (200 L): the liquid volume was 70%, and the secondary seed liquid of Enterobacter cancerogenus was inoculated in the fermentation tank for tertiary fermentation, and the inoculation amount was 5%. The culture temperature was 33°C, the shaking culture was performed at 200 rpm for 48 h. The formula of the medium used in the tertiary fermentation is as follows: 25 g / L of glucose, 14 g / L of potato juice 21 g / L of sucrose, 18 g / L of yeast powder, 3 g / L of magnesium sulfate, 4 g / L of disodium hydrogen phosphate, 4 g / L of NaCl, and pH 7. The effective viable count of Enterobacter cancerogenus SL12 obtained by fermentation was 8 billion CFU / mL. After the fermentation in the production tank was completed, the culture liquid was directly packaged into a liquid dosage form, which was a microbial inoculant containing Enterobacter cancerogenus SL12.

[0050] Example 4 Application of microbial inoculant

[0051] XY18 pepper seeds were selected for germination and seedling raising. Two treatments of microbial inoculant and CK (water) were set. The concentration of the microbial inoculant containing Enterobacter cancerogenus SL12 was adjusted to 1.0 x 10 8 CFU / mL.

[0052] At the eight-leaf stage of the XY18 pepper seedlings, the seedlings were transplanted into flowerpots (15 cm x 15 cm) filled with an equal amount of sterilized substrate nutrient soil. After 20 mL of microbial inoculant was poured, the seedlings were transplanted into the flowerpots, and 10 mL of SL12 inoculant was used for root irrigation treatment as a rooting water.

[0053] After 5 days of seedling, the pepper was treated by spraying and root irrigation at the same time, and the microbial inoculum was 20 mL, repeated 3 times. During the flowering period of pepper, spraying treatment was carried out every 2 days, and the microbial inoculum was 10 mL. The CK group used water instead of microbial inoculum, and the application time and amount were consistent with the microbial inoculum treatment group, and all the potting water and fertilizer management were the same.

[0054] During the fruiting stage, mature green fruits were collected uniformly, and mature green fruits were collected for each treatment, 5 peppers were selected for each repeat, the stem and seed in the pepper fruit were removed, and after drying at 50℃, the capsaicin content in the pepper fruit was determined by GB / T 21266-2007, and the results are shown in Table 1.

[0055] Table 1 Effect of microbial inoculum on capsaicin content in XY18 pepper fruit

[0056]

[0057] Table 1 is the effect of Enterobacter cancerogenus SL12 microbial inoculum treatment on the capsaicin content in XY18 pepper fruit. According to the table analysis, the average capsaicin content of XY18 fruit in the microbial inoculum treatment group was 130.3 mg / g, while the average capsaicin content of XY18 fruit in the water treatment was 80.5 mg / g. It can be seen that Enterobacter cancerogenus SL12 microbial inoculum can significantly increase the capsaicin content in pepper fruit, which is 61.9% higher than CK (water). Figure 4 The capsaicin content in the pepper fruit treated by Enterobacter cancerogenus SL12 microbial inoculum and water control is shown in the column chart, and "*" indicates that there is a significant difference between the treatments (P<0.05).

Claims

1. The application of a microbial inoculant in increasing the capsaicin content in chili pepper fruits, characterized in that, The microbial inoculant includes Enterobacter cancerogenus SL12 and its fermentation broth; the Enterobacter cancerogenus SL12 was deposited at the China Center for Type Culture Collection on March 25, 2022, with accession number CCTCCNO: M 2022288.

2. The application according to claim 1, characterized in that, The fermentation broth of Enterobacter SL12 is the unconcentrated original fermentation broth of Enterobacter SL12, and the effective viable count of the unconcentrated original fermentation broth of Enterobacter SL12 is 60 to 100 billion CFU / mL.

3. The application according to claim 1, characterized in that, The preparation method of microbial inoculants includes the following steps: S1. Enterobacter SL12 carcinogenic bacteria were inoculated into LB solid medium for activation to obtain activated Enterobacter SL12 carcinogenic bacteria; S2. The activated Enterobacter SL12 was inoculated into LB liquid medium for primary fermentation to obtain the primary seed culture of Enterobacter SL12; S3. The primary seed culture of Enterobacter SL12 was inoculated into the culture medium for secondary fermentation to obtain the secondary seed culture of Enterobacter SL12. S4. The secondary seed culture of Enterobacter carcinogen SL12 is inoculated into the culture medium for tertiary fermentation to obtain the microbial agent.

4. The application according to claim 3, characterized in that, In step S1, the activation conditions for *Enterobacter carcinogens* SL12 are: temperature 28-35℃, time 1-3 days; in step S2, the primary fermentation conditions are: liquid volume 40%-60%, inoculum 1%-3%, culture temperature 28-35℃, shaking culture at 180-200 rpm for 16-20 hours; in step S3, the secondary fermentation conditions are: liquid volume 50%-70%, inoculum 4%-7%, culture temperature 28-35℃, shaking culture at 180-200 rpm for 16-20 hours; in step S3, the volume ratio of the primary, secondary, and tertiary fermentation tanks is 1:20:200; in step S4, the tertiary fermentation conditions are: liquid volume 50%-70%, inoculum 4%-7%, culture temperature 28-35℃, shaking culture at 180-200 rpm for 36-48 hours.

Images