CN113957024A - Rhizobium GL-1803 and application thereof in preparation of insoluble beta-glucan - Google Patents
Rhizobium GL-1803 and application thereof in preparation of insoluble beta-glucan Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and particularly relates to rhizobium GL-1803 and application thereof in preparation of insoluble beta-glucan. The rhizobium GL-1803 has a preservation number of: CGMCC No. 23416. The new rhizobium GL-1803 is obtained by screening, the strain can convert sucrose into beta-1, 3-glucan, the conversion rate is up to more than 70%, and the obtained beta-1, 3-glucan is insoluble beta-1, 3-glucan.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to rhizobium GL-1803 and application thereof in preparation of insoluble beta-glucan.
Background
Beta-1, 3-glucan is a polysaccharide with a main chain formed by connecting beta-1, 3-glycosidic bonds, is a natural and harmless high molecular substance, and generally contains beta-1, 2-glycosidic bonds, beta-1, 4-glycosidic bonds and beta-1, 6-glycosidic bond connected branched chains in different proportions and sizes due to different sources.
The beta-1, 3-glucan has the biological activities of enhancing the immune activity, resisting tumors, oxidation, bacteria, viruses, fungi, cholesterol and blood fat, and the like, and is a good biological effect regulator. Meanwhile, the skin care product has the effects of moisturizing, resisting inflammation, resisting aging, removing wrinkles, removing dandruff, removing jaundice, accelerating repair, increasing skin elasticity and the like, and is widely applied to a plurality of fields of medicines, foods, cosmetics, animal feed additives and the like.
At present, the beta-1, 3-glucan is mainly derived from yeast, edible fungi and plants (such as shiitake mushroom, oat and highland barley) and is limited by the complexity of the technology and the high cost, the beta-1, 3-glucan is mainly a crude product with low purity, the production process generates more byproducts, and the yield is limited by the cost of raw materials. The beta-1, 3-glucan is produced by microbial fermentation, and has the advantages of no limitation of seasons, stable and easily obtained raw material sources, no byproduct generation in the process and stable quality among batches.
Therefore, it is necessary to develop a screening research work for efficiently producing beta-1, 3-glucan strains. Chinese patent application CN106434495A discloses a Bodhisattva rhizobium with the preservation number of CGMCC No.12954, which is fermented to produce water-soluble beta-1, 3-glucan; chinese patent application CN112358985A discloses a strain of Przewalsk rhizobium with the preservation number of CGMCC No.19764, and the strain is also fermented to produce water-soluble beta-1, 3-glucan.
Disclosure of Invention
In view of the above problems, the main object of the present invention is to provide a novel strain capable of producing beta-1, 3-glucan at a high yield.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a rhizobium GL-1803 strain, which is prepared from (A) rhizobiumRhizobiumsp.) GL-1803 was deposited at the China general microbiological culture Collection center on the 09.15.2021 year old with the following deposit numbers: CGMCC No.23416, preservation Address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
The strain GL-1830 for producing beta-1, 3-glucan at high yield is obtained by screening a soil sample collected from the position 10cm below the ground surface of humid soil and grass in the Jinan yellow river wetland park, wherein the soil sample is adjacent to humus at the root of a tree, and the strain GL-1803 can convert sucrose into water-insoluble beta-1, 3-glucan with the conversion rate of over 70 percent. The strain is identified:
morphological identification: GL-bacterial colony is circular, the rod shaped bacterium is white, the edge is neat, the GL-bacterial colony is moist and viscous, and the GL-bacterial colony is rich in elasticity.
Physiological and biochemical identification: gram-negative bacteria.
Amplification and sequencing of 16 SrDNA: the sequence of the strain GL-183016 SrDNA is shown in SEQ ID No. 1. The 16SrDNA sequence obtained by sequencing is aligned with BLAST in GenBank and has 100 percent of homology with the rhizobium related fragment sequence published in GenBank. The strain GL-1803 of the invention is identified as rhizobium (Rhizobium) (by combining the results of physiological and biochemical identification and 16S rRNA sequence analysis and identificationRhizobium sp.)。
The invention also provides a rhizobium GL-1803 fermentation medium which comprises the following components: sucrose 5.0% (by mass), (NH)4)2HPO4 0.1%～1.0%，KH2PO4 0.15%～0.25%，MgSO4•7H2O 0.1%，CaCO30.15 to 0.3 percent of corn steep liquor, 0.1 to 0.5 percent of corn steep liquor powder and the balance of water, and the pH value is adjusted to 7.0.
The invention also provides application of rhizobium GL-1803 in preparation of insoluble beta-glucan. The rhizobium GL-1803 disclosed by the invention not only can be used for producing beta-1, 3-glucan at a high yield, but also can be used for obtaining insoluble beta-1, 3-glucan.
Compared with the prior art, the invention has the following advantages:
the invention obtains a new rhizobium GL-1803 strain through screening, the strain can convert sucrose into beta-1, 3-glucan, and the conversion rate is up to more than 70%. The beta-1, 3-glucan obtained by the fermentation of the strain is insoluble in water and is insoluble beta-1, 3-glucan. The invention further enriches the types of the beta-1, 3-glucan produced by microbial fermentation.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, 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 belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular is intended to include the plural unless the context clearly dictates otherwise, and it should be understood that the terms "comprises" and "comprising," when used in this specification, specify the presence of the stated features, steps, operations, and combinations thereof.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
Screening and identification of Rhizobium GL-1803:
(I) screening of the strain GL-1803:
step one, enrichment: collecting soil samples from humid soil and grass in the Jinan yellow river wetland park at a position 10cm below the ground surface of humus near the root of a tree, collecting the soil samples, placing the collected soil samples into an oven for drying, sieving the dried soil with a 100-mesh sieve, accurately weighing 1g of the soil sieved with the 100-mesh sieve in 9mL of sterile water in an aseptic manner, standing for 30min, oscillating for 30min in a shaking table at room temperature to obtain the dilution of 10-1The soil suspension of (a); sucking out 1mL of the above soil suspension, adding 9mL of sterile water, and mixing to obtain a dilution of 10-2The soil suspension of (a); from a dilution of 10-2Sucking out 1mL of the soil suspension, adding 9mL of sterile water, and mixing to obtain a dilution of 10-3The soil suspension of (a);
step two, primary screening and purification: taking the dilution degree in the step one as 10-30.1mL of the soil suspension is coated on aniline blue screening medium by a coating plate method, and is placed in a constant temperature incubator at 28 ℃ for inverted culture for 5 daysTaking the bacterial strain with the blue circle around the bacterial colony as the bacterial strain with the beta-1, 3-glucan production capacity, selecting the bacterial strain with the blue circle around the bacterial colony, carrying out multiple streaking separation and purification on an aniline blue screening culture medium to obtain purified bacterial strains (10 strains in total) with consistent forms through purification, transferring the purified bacterial strains to a nutrient agar culture medium, and storing the purified bacterial strains in a refrigerator at 4 ℃ for later use; respectively marking the stored strains as GL1801-GL 1810; the aniline blue culture medium comprises the following components: 20g of sucrose, 3g of yeast extract, 5g of peptone, 2g of sodium chloride, 1g of aniline blue and 20g of agar powder, distilled water is added to the solution to reach 1000mL, and the pH value of a culture medium is 7.0; the nutrient agar culture medium comprises the following components: 20g of sucrose, 3g of yeast extract, 5g of peptone, 2g of sodium chloride and 20g of agar powder, distilled water is added to the mixture until the volume is 1000mL, and the pH value of the culture medium is 7.0.
Step three, re-screening: activating the strain stored in the step two to obtain a bacterial suspension, dripping the bacterial suspension on an aniline blue screening culture medium, culturing for 3 days at 30 ℃, measuring the diameter D of a blue ring generated by the strain and the diameter D of a bacterial colony by taking 1 mu L of sterile water as a reference, and preliminarily determining the beta-1, 3-glucan producing capacity of the strain according to the D/D value, wherein the result is shown in Table 1.
TABLE 1 determination of beta-1, 3-glucan-producing ability of 10 strains by color circle method
| Diameter D/mm of blue circle | Diameter d/mm of bacterial colony | Ratio D/D | |
| GL-1801 strain | 26 | 24 | 1.08 |
| GL-1802 strain | 26 | 25 | 1.04 |
| GL-1803 strain | 28 | 24 | 1.17 |
| GL-1804 strain | 25 | 24 | 1.04 |
| GL-1805 strain | 26 | 25 | 1.04 |
| GL-1806 strain | 27 | 26 | 1.04 |
| GL-1807 strain | 27 | 24 | 1.13 |
| GL-1808 strain | 27 | 25 | 1.08 |
| GL-1809 bacteriumPlant strain | 27 | 24 | 1.13 |
| GL-1810 Strain | 26 | 24 | 1.08 |
| Sterile water | 0 | 0 | 0 |
As can be seen from Table 1, the blue circle of GL-1803 strain is the largest among the 10 strains with the ability to produce beta-1, 3-glucan obtained by screening, and the D/D value reaches 1.17, so that the GL-1830 strain has the strongest ability to produce beta-1, 3-glucan.
(II) identification of Strain GL-1803
(1) Morphological identification: activating the screened GL-1803 strain on a culture medium, and observing the growth characteristics of bacterial colonies; single colonies were picked with the inoculating loop for gram staining, microscopic examination, and results recorded.
As a result: GL-bacterial colony is circular, the rod shaped bacterium is white, the edge is neat, the GL-bacterial colony is moist and viscous, and the GL-bacterial colony is rich in elasticity.
(2) Physiological and biochemical identification: gram-negative bacteria.
(3) Amplification and sequencing of 16 SrDNA:
the identification of the strain is completed by the institute of microorganisms of Chinese academy of sciences.
The identification process of the strain comprises the steps of extracting strain DNA, carrying out PCR amplification, carrying out gel electrophoresis, purifying and recovering, connecting, preparing competent cells, converting a connecting product, extracting and sequencing plasmids, and comparing a purified 16S rRNA sequence on a ribosome database; wherein the primer sequence of PCR amplification is as follows:
an upstream primer: 5 '-AGTTTGATCMTGGCTCAG-3'
A downstream primer: 5'-GGTTACCTTGTTACGACTT-3'
In the reaction system of PCR amplification, the addition amount of each substance is as follows: 0.5 microliter of template, wherein the concentration of the genome DNA is 20 ng/nL-50 ng/nL; 2.5 μ L of 10 × buffer, 1 μ L of dNTP with the concentration of 2.5mM, 0.2 μ L of enzyme, 1 μ L of each F with the concentration of 10 μ M, and 1 μ L of R with the concentration of 10 μ M, and adding double distilled water to make the volume to be 25 μ L; and (3) amplification procedure: pre-denaturation at 94 ℃ for 4min and denaturation at 94 ℃ for 45 s; annealing at 55 ℃ for 45 s; extending for 1min at 72 ℃; after 30 cycles, the extension was carried out at 72 ℃ for 10min and the reaction was stopped at 4 ℃.
GL-1803 Strain 16SrDNA sequence analysis and alignment: the fragment size of the PCR amplified product 1403pb, and the sequence of strain GL-183016 SrDNA is shown in SEQ ID No. 1. BLAST alignment of the sequenced 16SrDNA sequence in GenBank is carried out, and the sequence has 100 percent homology with the sequence of related rhizobium fragments published in GenBank. The strain GL-1803 of the invention is identified as rhizobium (Rhizobium) (by combining the results of physiological and biochemical identification and 16S rRNA sequence analysis and identificationRhizobium sp.）。
Example 2
Adding 100mL of fermentation medium into a 500mL triangular flask, sterilizing at high temperature and high pressure, inoculating GL-1803 strain into the fermentation medium according to the proportion of 5%, performing shake culture at 30 ℃ and 220r/min for 5 days, centrifuging in a centrifuge at the rotation speed of 8000r/min for 20 minutes, adding 100mL of purified water into the centrifuged precipitate, mixing uniformly, centrifuging the uniformly mixed suspension in the centrifuge at the rotation speed of 8000r/min for 20 minutes, drying the centrifuged precipitate in an oven at the temperature of 45 ℃, drying and grinding into powder. Putting 0.1000g of powder into a 100mL volumetric flask, adding about 90mL of 0.1mol/L sodium hydroxide solution, shaking up to dissolve, adding 0.1mol/L sodium hydroxide solution to fix the volume to 100mL, putting 5.0mL of solution into the 100mL volumetric flask, adding purified water to fix the volume to 100mL, putting 1.0mL of diluent into a colorimetric tube, adding 1.0mL of 5% phenol solution, adding 5.0mL of concentrated sulfuric acid, mixing uniformly, cooling to the normal temperature, taking 0.1mL of purified water as a blank, taking dry glucose powder as a control, measuring absorbance at 490nm and determining the total glucose amount.
The fermentation medium comprises the following components in percentage by mass: 5% of sucrose (NH)4)2HPO4 0.2%，KH2PO40.2%，MgSO4•7H2O 0.1%，CaCO30.2 percent of corn steep liquor powder, 0.2 percent of distilled water and the balance of the distilled water, wherein the pH value of the culture medium is 7.0.
The GL-1803 strain converted sucrose to water-insoluble beta-1, 3-glucan with a conversion of 72.08%.
Example 3
The difference compared to example 2 is that the composition of the fermentation medium is: the weight percentage of the material is as follows: 5% of sucrose (NH)4)2HPO4 0.22%，KH2PO4 0.2%，MgSO4•7H2O 0.1%，CaCO30.2 percent of corn steep liquor powder, 0.2 percent of distilled water and the balance of the distilled water, wherein the pH value of the culture medium is 7.0. The other steps were the same as in example 2.
The GL-1803 strain converted sucrose to water-insoluble beta-1, 3-glucan at 71.93%.
Example 4
The difference compared to example 2 is that the composition of the fermentation medium is: the composition comprises the following components in percentage by mass: 5% of sucrose (NH)4)2HPO4 0.2%，KH2PO4 0.22%，MgSO4•7H2O 0.1%，CaCO30.2 percent of corn steep liquor powder, 0.2 percent of distilled water and the balance of the distilled water, wherein the pH value of the culture medium is 7.0. The other steps were the same as in example 2.
The GL-1803 strain converted sucrose to water-insoluble beta-1, 3-glucan at 72.09%.
Examples 2-4 the results of the measurement of β -1, 3-glucan production by GL-1803 strains are shown in Table 2 below.
TABLE 2 measurement results of beta-1, 3-glucan production by GL-1803 strain
| Sucrose content, g/L | Crude glucan yield, g/L | Absorbance at 490nm | Glucan content% | Conversion rate% | |
| Control | 0.509 | 100 | |||
| Example 2 | 50 | 44.9 | 0.454 | 80.27 | 72.08 |
| Example 3 | 50 | 45.1 | 0.451 | 79.74 | 71.93 |
| Practice ofExample 4 | 50 | 45.4 | 0.449 | 79.39 | 72.09 |
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Shandong national force Biotechnology GmbH, Shandong national force Biotechnology research institute
<120> rhizobium GL-1803 and application thereof in preparation of insoluble beta-glucan
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1353
<212> DNA
<213> Artificial sequence ()
<400> 1
ggttagctgc ctccttgcgg ttagcgcact accttcgggt aaaccaactc ccatgggtgt 60
gacgggcggt gtgtacaagg cccgggaacg tattcaccgc agcatgctga tctgcgatta 120
ctagcgattc caacttcatg cactcgagtt gcagagtgca atccgaactg agatggcttt 180
tggagattag ctcgacatcg ctgtctcgct gcccactgtc accaccattg tagcacgtgt 240
gtagcccagc ccgtaagggc catgaggact tgacgtcatc cccaccttcc tctcggctta 300
tcaccggcag tccccttaga gtgcccaact aaatgctggc aactaagggc gagggttgcg 360
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacagcc atgcagcacc 420
tgttctgggg ccagcctaac tgaaggacat cgtctccaat gcccataccc cgaatgtcaa 480
gagctggtaa ggttctgcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540
gcccccgtca attcctttga gttttaatct tgcgaccgta ctccccaggc ggaatgttta 600
atgcgttagc tgcgccaccg aacagtatac tgcccgacgg ctaacattca tcgttacggc 660
gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgcacct cagcgtcagt 720
aatggaccag taagccgcct tcgccactgg tgttcctccg aatatctacg aatttcacct 780
ctacactcgg aattccactt acctcttcca tactcaagat acccagtatc aaaggcagtt 840
ccgcagttga gctgcgggat ttcacccctg acttaaatat ccgcctacgt gcgctttacg 900
cccagtaatt ccgaacaacg ctagccccct tcgtattacc gcggctgctg gcacgaagtt 960
agccggggct tcttctccga ctaccgtcat tatcttcatc ggtgaaagag ctttacaacc 1020
ctaaggcctt catcactcac gcggcatggc tggatcaggc ttgcgcccat tgtccaatat 1080
tccccactgc tgcctcccgt aggagtttgg gccgtgtctc agtcccaatg tggctgatca 1140
tcctctcaga ccagctatgg atcgtcgcct tggtaggcct ttaccccacc aactagctaa 1200
tccaacgcgg gctcatccat ccccgataat ctttcccccg tagggcgtat gcggtattaa 1260
ttccagtttc ccagagctat tccgcagaga tgggtagatt cccacgcgtt actcacccgt 1320
ctgccactcc ccttgcgggg cgttcgactc gca 1353
Claims (3)
1. A rhizobium GL-1803 strain is characterized in that rhizobium (A)Rhizobiumsp.) GL-1803 was deposited at 15.09.2021 in the China general microbiological culture Collection center (CGMCC)The preservation number is: CGMCC number 23416, storage address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
2. The rhizobia GL-1803 fermentation medium of claim 1, wherein the fermentation medium comprises the following components: sucrose 5.0% (by mass), (NH)4)2HPO4 0.1%～1.0%，KH2PO4 0.15%～0.25%，MgSO4•7H2O 0.1%，CaCO30.15 to 0.3 percent of corn steep liquor, 0.1 to 0.5 percent of corn steep liquor powder and the balance of water, and the pH value is adjusted to 7.0.
3. Use of rhizobia GL-1803 as defined in claim 1 for the preparation of insoluble β -glucan.
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