CN112980720B - Enterobacter chengduensis LB-132 strain and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microbiology, and particularly relates to an Enterobacter chengduensis LB-132 strain and application thereof. An Enterobacter chegduensis LB-132 strain is classified as Enterobacter gondoensis, is preserved in China general microbiological culture Collection center on 9-14 th month 2020, and has a preservation number of CGMCC No. 20650. The LB-132 strain can convert ginsenoside Rg into ginsenoside Rg₁Can be rapidly converted into ginsenoside F₁In a constant temperature incubator at 28 ℃ for 160 r.min^‑1After 10 days of culture, the average conversion rate can reach 13.24 percent, an ideal way is provided for obtaining rare ginsenoside, and simultaneously, materials can be provided for developing special fertilizers or microbial inocula for ginseng.

Description

Enterobacter chengduensis LB-132 strain and application thereof

Technical Field

The invention belongs to the technical field of microbiology, and particularly relates to an Enterobacter chengduensis LB-132 strain and application thereof.

Background

Dried ginseng belonging to perennial herbaceous plantsThe root and the rhizome are dried, the pharmacological action is wide, and the medicinal value and the application prospect are extremely high. The saponin is the main effective component of Panax ginseng Panaxginseng and Panax notoginseng Panaxnogening, and has effects of resisting tumor, resisting aging, softening blood vessel, and protecting liver. Rare ginsenoside F₁Has antitumor, antioxidant, and antiinflammatory pharmacological activities, and is rare ginsenoside F₁The content of the ginsenoside in natural panax plants is very small, so the research of converting the ginsenoside into the rare ginsenoside F1 has important significance.

In recent years, the methods for performing biotransformation using ginsenoside as a raw material mainly include chemical methods and biological methods, and among them, the biotransformation method has the advantages of high specificity, low cost, mild reaction conditions, high selectivity, environmental friendliness, and the like, and is considered to be the most effective method for obtaining rare ginsenoside. Endophytes (Endophytes) refer to a group of microorganisms, mainly including fungi, bacteria and actinomycetes, that survive within healthy plant tissues without causing the host plant to exhibit significant symptoms of infection. In recent years, the use of endophytes for bioconversion has attracted a great deal of attention and has been applied to the biosynthesis of some natural compounds such as flavans, tetrahydroxylopyranosides and astragalosides. Therefore, the screening and culture of the bacterial strain capable of converting the ginsenoside into the rare ginsenoside from the plant endophyte have important significance.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide an Enterobacter chengduensis LB-132 strain and application thereof.

The Enterobacter chengduensis LB-132 strain provided by the invention is separated from pseudo-ginseng, is identified as Enterobacter donovani (Enterobacter chengduensis) by morphology and molecular biology, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 9-14 th 2020 (CGMCC for short, the address: Beijing City & Onyang district No. 1 Beichen West Lu No.3, the institute of microorganisms of China academy of sciences, zip code: 100101), and has the preservation number of CGMCC No. 20650.

The invention also provides the application of the Enterobacter chengduensis LB-132 strain in preparing ginsenoside Rg₁Conversion to ginsenoside F₁The use of (1).

Compared with the prior art, the invention has the following beneficial technical effects:

the LB-132 strain can convert ginsenoside Rg into ginsenoside Rg₁Can be rapidly converted into ginsenoside F₁In a constant temperature incubator at 28 ℃ for 160 r.min^-1After 10 days of culture, the average conversion rate can reach 13.24 percent, an ideal way is provided for obtaining rare ginsenoside, and simultaneously, materials can be provided for developing special fertilizers or microbial inocula for ginseng.

Description of preservation information

Enterobacter medoxomil (Enterobacter chegduensis) LB-132 with the preservation number of CGMCC No.20650, the preservation date of 2020, 9 and 14 days, the preservation unit is China general microbiological culture Collection center (CGMCC), and the preservation address is No.3 of Beijing Shang-Kogyang Beichen No. 1 Kte.

Drawings

FIG. 1 is an electron microstructure of a strain of LB-132 of the present invention;

FIG. 2 is a tree of evolutionary trees of the resulting sequences constructed;

FIG. 3 is an HPLC chart of LB-132 of the present invention;

description of the reference numerals:

a is an HPLC chart of a ginsenoside standard substance, wherein 1-5 are respectively: ginsenoside Rg₁、Rb₁、F₁Rd and Rg₃。

b is ginsenoside Rg₁And an HPLC profile of sterile liquid medium;

c is ginsenoside Rg₁Inoculated with the HPLC chart of LB-132 after culture.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The materials and reagents used in the following examples are commercially available, unless otherwise specified. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The standards, reagents and instruments used in the following examples are as follows:

and (3) standard substance: accurately weighing reference ginsenoside Rg respectively, drying under reduced pressure to constant weight₁、Rb₁、Rd、Rg₃、F₁10mg, placing the mixture into a 1mL brown volumetric flask, adding methanol for dissolution, fixing the volume and placing scales, wherein the mass concentration is 10 mg/mL.

Experimental reagent: FastDNA Spin Kit for Soil Kit (MoBio Laboratories, Inc., USA), absolute ethanol (Tech technologies, Inc., Tianjin), sodium hypochlorite (Tianjin Mada, Inc.), and 27F/338R amplification primers (Shanghai Biotech, Inc.).

An experimental instrument: high performance liquid chromatograph (1260, Agilent), sterilization pan (SN51OC, chongqing yamaoto technologies ltd.), electronic balance (ME203E/02, mettler-toledo instruments ltd.), homogenizer (MP FastPrep-245G, american MP), vortexer (ABSON, MixStat), centrifuge (SIGMA, Germany), PCR instrument (2720, hangzhou gathers ltd.), electrophoresis instrument (DYY-8C, beijing six instruments factory), microscope (COSSI, olympson), electrophoresis gel imager (JY04S-3C, beijing sovereign).

Example 1: endophyte separation, purification and identification

1.1 isolation and purification of endophytes

Taking fresh and healthy roots, stems and leaves of pseudo-ginseng, washing with running water, performing surface disinfection treatment, cutting the tissues into 0.5cm in size by using a sterile scalpel, inoculating the cut tissues into an LB (Luria Broth) culture medium, culturing the cut tissues at 37 ℃ until bacterial plaques grow out, respectively picking different bacterial colonies into the LB culture medium, and gradually separating and purifying until a purified single bacterial strain is obtained.

1.2 morphological identification

Picking single bacteria with different forms to make temporary loading pieces, and observing colony forms under an electron microscope, wherein the electron microscopic structure of the strain of LB-132 is shown in figure 1.

As can be seen in FIG. 1, LB-132 is in the form of a straight rod.

1.3 molecular characterization

The gene kit is adopted for extraction, 16S universal primers in the following table 1 are used for sequencing and identification, and the sequencing result is shown as SEQ ID No. 3.

Table 116S Universal primers

Constructing an evolutionary tree of the resulting sequence, as shown in FIG. 2; the evolutionary tree is combined, online homology comparison is carried out on the obtained sequence, the similarity of the strain and the Enterobacter chengduensis is found to be higher, the separated strain is determined to be the Enterobacter chengduensis, and the strain is named as a strain LB-132.

Example 2: application of strain

Adding 1900 μ L sterile PDA liquid culture medium and 100 μ L10 mg/mL ginsenoside Rg into 5mL centrifuge tube₁Then inoculating 2mL strain LB-132, culturing at 28 deg.C constant temperature incubator for 10d at 160r/min, adding n-butanol with solubility of 600 μ L to dissolve, stopping reaction, centrifuging at high speed, collecting supernatant, performing HPLC analysis, and calculating ginsenoside F for 3 times of repetition₁The conversion of (a); HPLC chart is shown in figure 3, ginsenoside F₁The conversion of (d) is shown in table 2 below.

TABLE 2 LB-132 ginsenoside F₁Conversion ratio of (1%)

As can be seen from FIG. 3 and Table 2, the ginsenoside RG can be extracted from LB-132 of the present invention₁Conversion to ginsenoside F₁The average conversion of 3 replicates was 13.24%.

In conclusion, the LB-132 strain of the invention can be used for preparing ginseng soapGlycoside Rg₁Can be rapidly converted into ginsenoside F₁Provides an ideal way for obtaining rare ginsenoside, and simultaneously provides a material for developing special fertilizer or microbial inoculum for ginseng.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Sequence listing

<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine

<120> Enterobacter chengduensis LB-132 strain and application thereof

<130> 2020

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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agagtttgat cctggctcag 20

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tgctgcctcc cgtaggagt 19

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<211> 1393

<212> DNA

<213> Enterobacter chengduensis (Enterobacter chengduensis)

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tcccgaaggt taagctacct acttcttttg caacccactc ccatggtgtg acgggcggtg 60

tgtacaaggc ccgggaacgt attcaccgta gcattctgat ctacgattac tagcgattcc 120

gacttcatgg agtcgagttg cagactccaa tccggactac gacgcacttt atgaggtccg 180

cttgctctcg cgaggtcgct tctctttgta tgcgccattg tagcacgtgt gtagccctac 240

tcgtaagggc catgatgact tgacgtcatc cccaccttcc tccagtttat cactggcagt 300

ctcctttgag ttcccggcct aaccgctggc aacaaaggat aagggttgcg ctcgttgcgg 360

gacttaaccc aacatttcac aacacgagct gacgacagcc atgcagcacc tgtctcagag 420

ttcccgaagg caccaatcca tctctggaaa gttctctgga tgtcaagagt aggtaaggtt 480

cttcgcgttg catcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc 540

atttgagttt taaccttgcg gccgtactcc ccaggcggtc gacttaacgc gttagctccg 600

gaagccacgc ctcaagggca caacctccaa gtcgacatcg tttacggcgt ggactaccag 660

ggtatctaat cctgtttgct ccccacgctt tcgcacctga gcgtcagtct ttgtccaggg 720

ggccgccttc gccaccggta ttcctccaga tctctacgca tttcaccgct acacctggaa 780

ttctaccccc ctctacaaga ctctagcctg ccagtttcga atgcagttcc caggttgagc 840

ccggggattt cacatccgac ttgacagacc gcctgcgtgc gctttacgcc cagtaattcc 900

gattaacgct tgcccctccg tattaccgcg gctgctggca cggagttagc cggtgcttct 960

tctgcgggta acgtcaattg ctgaggttat taacctcaac accttcctcc ccgctgaaag 1020

tactttacaa cccgaaggcc ttcttcatac acgcggcatg gctgcatcag gcttgcgccc 1080

attgtgcaat attccccact gctgcctccc gtaggagtct ggaccgtgtc tcagttccag 1140

tgtggctggt catcctctca gaccagctag ggatcgtcgc ctaggtgagc cgttacccca 1200

cctactagct aatcccatct gggcacatct gatggcaaga ggccgaaggt ccccctcttt 1260

ggtcttgcga cgttatgcgg tattagctac cgtttccagt agttatcccc ctccatcagg 1320

cagtttccca gacattactc acccgtccgc cgctcgtcac ccaggagcaa gctccctgtg 1380

ttaccgctcg act 1393

Claims (2)

1. An Enterobacter chegduensis LB-132 strain is classified as Enterobacter gondoensis, is preserved in China general microbiological culture Collection center on 9-14 th month 2020, and has a preservation number of CGMCC No. 20650.

2. The Enterobacter chegduensis LB-132 strain of claim 1, wherein ginsenoside Rg is extracted from the strain₁Conversion to ginsenoside F₁The use of (1).

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