CN109777752B - Polysaccharide-producing psychrobacter pneumococcus T-15 strain and application thereof - Google Patents

Abstract

The invention provides a T-15 strain for producing polysaccharide, belonging to a Psychrobacter pneumophilus (Psychrobacter pulmonis strain), which is preserved in China center for type culture collection with the address: china, wuhan university; the zip code 430072 has a preservation date of 2019, 1 month and 17 days, and has a preservation number of: CCTCC M2019056; the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1. The psychrobacter pneumococcus T-15 strain can be used for preparing polysaccharide by fermentation. The strain provided by the invention can be subjected to large-scale fermentation culture in a short period, has low fermentation cost and is not limited by conditions such as regions, seasons and the like, so that the current situation that the natural resources of water shield are increasingly deficient can be solved through a microbial fermentation way, and higher economic benefit is realized.

Description

Polysaccharide-producing psychrobacter pneumococcus T-15 strain and application thereof

Technical Field

The invention relates to a psychrophile pneumococcus strain which can produce polysaccharide, belonging to the technical field of microorganisms.

Background

At present, the research of plant endophytes is gradually paid attention, and various endophytes of different plants are discovered in succession. The diversity of endophyte species also endows the endophyte with the diversity of metabolites, which also indicates that the exploitation of new resources such as biology, chemistry, medicine and the like by utilizing the metabolism of microorganisms has great potential. A large number of researches show that the plant endophyte can produce active substances and precursors which are the same as or similar to those of a host plant, and the discovery directly promotes the wide development of the research of the plant endophyte, particularly the economic plant endophyte. With the gradual research of people on the economic plant endophytes, the endophytes capable of metabolizing to generate corresponding active ingredients are utilized to produce novel, efficient and low-cost active drugs, the current situation that rare economic plant resources are in short supply is solved, and the method is bound to become the key point of the future economic plant endophyte research.

Although endophytes, particularly economic endophytes, have received increasing attention from a large number of scholars in recent years, endophyte research on hundreds of plants is still being conducted. Brasenia schreberi (Brasenia schreberi) also called water chestnut, lake vegetable, water sunflower, dew sunflower, water lotus leaf, cauliflower and the like is a perennial leafy aquatic plant belonging to the genus Brasenia of the family Nymphaeaceae. The viscous pectin on the surface of the water shield is mainly composed of viscous polysaccharide, and has high nutritive value. Water shield is an important aquatic economic plant, and has high requirement on water quality. Under the situation that the wild environment of the water shield is increasingly deteriorated, finding an alternative way for producing the water shield polysaccharide is particularly important.

The brasenia schreberi endophyte has been concerned by people in recent years, and generally, researches on brasenia schreberi endophytes, particularly on brasenia schreberi polysaccharide components capable of being metabolized to generate corresponding brasenia schreberi polysaccharides, are still relatively few, and reports on brasenia schreberi endophytes for producing brasenia schreberi polysaccharides are rare.

Disclosure of Invention

The invention solves the problems in the background technology and provides a psychrobacter pulmonary tip T-15 strain for producing polysaccharide, and the strain can produce brasenia schreberi polysaccharide.

The inventor selects a novel strain, which is named as T-15 and belongs to psychrophilic bacillus (Psychrobacter pulmonis strain), and the strain is preserved in China center for type culture Collection, and the address is as follows: china, wuhan university; the zip code 430072 has a preservation date of 2019, 1 month and 17 days, and has a preservation number of: CCTCC M2019056; the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.

The psychrobacter pneumococcus T-15 strain can be used for preparing polysaccharide by fermentation.

The method for preparing the polysaccharide by using the psychrobacter pneumocystis T-15 strain comprises the following steps: firstly, inoculating a psychrophilum apicomplexa T-15 strain into an LB liquid culture medium, carrying out shaking culture at the rotating speed of 200r/min and the temperature of 37 ℃ overnight for 2 days, centrifuging a zymogen solution at 12000r/min, and taking a supernatant; mixing the supernatant with chloroform-n-butyl alcohol solution in a centrifuge tube, oscillating for 20-30min, centrifuging again, taking the supernatant, filtering the supernatant twice with a 0.45 mu m filter membrane, heating and concentrating at 70 ℃ to 1/2-2/3 of the original volume, adding 95% ethanol of 4 times of the original volume, putting the mixture into a refrigerator for freezing overnight, and finally centrifuging at 12000r/min for 10min to obtain the polysaccharide precipitate.

Compared with the prior art, the invention has the following advantages: the psychrobacter pneumococcus T-15 strain provided by the invention can be metabolized to produce polysaccharide. The polysaccharide produced by metabolism has the same HPLC spectral peak and similar polysaccharide infrared spectrum characteristic peak as well as similar or identical chemical functional groups as the polysaccharide separated and purified from natural water shield. Compared with natural water shield polysaccharide, the biological activity of the polysaccharide prepared by fermenting the strain is obviously higher than that of the natural water shield polysaccharide in hydroxyl radical scavenging effect, and the polysaccharide has better antioxidant activity.

The strain provided by the invention can be subjected to large-scale fermentation culture in a short period, has low fermentation cost and is not limited by conditions such as regions, seasons and the like, so that the current situation that the natural resources of water shield are increasingly deficient can be solved through a microbial fermentation way, and higher economic benefit is realized.

Drawings

FIG. 1 is a colony morphology diagram of the strain provided by the invention in a culture medium;

FIG. 2 is a High Performance Liquid Chromatography (HPLC) analysis chart of the water shield polysaccharide extract and the bacterial strain fermentation product polysaccharide provided by the invention;

FIG. 3 is an infrared spectrum analysis diagram of the polysaccharide of the strain fermentation product provided by the invention.

Detailed Description

The present invention will be described in detail with reference to specific examples, but the scope of the present invention is not limited to the examples.

Separation of water shield endophyte

Taking stems, leaves and roots of fresh water shield, sterilizing with mercuric chloride under aseptic environment, rinsing with distilled water for several times, inoculating onto LB solid culture medium, and culturing in dark place. Colonies that grew well were picked from the medium and transferred to a new LB medium plate, purified and numbered respectively. The strain provided in this example was named strain T-15, and colonies of this strain were transferred to a new LB medium plate and grew well, and the colony morphology thereof is shown in fig. 1, in which the scale bar in fig. 1 is 2 mm. As can be seen from FIG. 1, the diameter is about 3-5mm, and the colonies are relatively round and milky white.

Identification of water shield endophyte

After the separated and purified endophyte is processed (DNA extraction or boiling treatment), the bacterial 16S rDNA sequence is amplified (by using 27F/1492R primer), the amplification product is subjected to sanger sequencing, the sequencing result is compared and analyzed on an NCBI website (https:// blast.

16S rDNA sequence amplification primer sequence:

primer 127F: 5'-AGAGTTTGATCCTGGCTCAG-3'

Primer 21492R: 5'-TACGGCTACCTTGTTACGACTT-3'

The amplified fragment has a size of about 1400-1700bp

According to a standard sequencing method of bacteria, the sequence of the brasenia schreberi endophyte is shown as SEQ ID No.1, and the sequencing result is compared and analyzed on an NCBI website, and the result shows that the brasenia schreberi endophyte provided by the embodiment is the bacteria, and the Latin of the endophyte is named as Actinomyces pulmonarius (Psychrobacter pulmonis strain).

The applicant reserves the strain in China center for type culture Collection, address: china, wuhan university; the zip code 430072 has a preservation date of 2019, 1 month and 17 days, and the patent strain preservation numbers are as follows: CCTCC M2019056.

Preparation of water shield endophyte polysaccharide

Respectively picking part of the strains from the LB culture medium plate, transferring the strains to a purified strain, inoculating the strains into an LB liquid culture medium, carrying out shaking culture at the rotating speed of 200r/min and the temperature of 37 ℃ overnight for 2 days, centrifuging the zymogen liquid at 12000r/min for 5 minutes, and taking the supernatant. Preparing chloroform-n-butanol solution (chloroform: n-butanol is 4:1), mixing the supernatant with organic solution at a ratio of 3:1, centrifuging for 10min at 8000r/min under shaking for 20-30min, collecting supernatant, and collecting protein precipitate between two phases. Protein removal was continued for 5 times as described above. Filtering the supernatant twice with 0.45 μm filter membrane, heating at 70 deg.C, concentrating to 1/2-2/3, adding 4 times of 95% ethanol, freezing overnight in refrigerator, centrifuging at 12000r/min for 10min the next day, and precipitating to obtain endophyte polysaccharide.

In order to compare whether the fermentation liquor of the water shield endophyte provided by the invention contains polysaccharide, polysaccharide content determination is carried out on the endophyte fermentation liquor extract.

Firstly, crude polysaccharide is extracted and separated from natural water shield as a reference substance, and the method comprises the following steps: taking water shield tender shoots, putting the water shield tender shoots into NaOH (0.1mol/L) for leaching, wherein the material-liquid ratio is 1:2(w/v), leaching with alkali liquor at 25 ℃ for 1.5h, and adjusting the pH value to 7.0. Picking out water shield bud, centrifuging the residue with centrifuge, and concentrating the supernatant with magnetic stirrer at 60 deg.C for 1 hr. The concentrated crude extract was then repeatedly treated by the sevage method to remove free proteins and was again concentrated to about 2/3 of the original volume. Adding four times volume of anhydrous ethanol for precipitation overnight, centrifuging the mixture at 4000r/min for 10min to obtain precipitate, dissolving the precipitate with small amount of water, concentrating, adding four times volume of ethanol again for precipitation overnight, repeating the operation three times, and drying to obtain water shield polysaccharide (BSP).

The measurement method is as follows: after polysaccharide is hydrolyzed by sulfuric acid, a DNS method (3-amino-5-nitro salicylic acid) is adopted to determine the content of secreted sugar in the bacterial liquid, D-glucuronic acid is taken as a standard, and a standard curve is firstly made through the absorbance of glucose standard liquid with different concentrations after the glucose standard liquid reacts with a DNS reagent and sulfuric acid. The standard curve is determined to be y-0.0368 x-0.0436 (R)²0.9807). The polysaccharide content was determined according to a standard curve. Experimental results show that the content of polysaccharide in the BSP sample is 0.30 +/-0.01 mg/mL, and the content of polysaccharide in the fermentation extract of the T-15 strain of the Acinetobacter pneumocystus is 0.06 +/-0.001 mg/mL.

Characterization of polysaccharide Components

To compare whether the Brasenia schreberi endophyte polysaccharide and Brasenia schreberi polysaccharide have the same polysaccharide components, HPLC analysis was performed in this example. The results are shown in fig. 2, wherein the upper graph is the endophyte polysaccharide in the example, and the lower graph is the extracted natural Brasenia schreberi polysaccharide, it can be seen from fig. 2 that the extract of the polysaccharide from the actinomycete belonging to the genus Acidophilus at the time of 19min has a peak, and a secondary peak appears at the time of 21min, and it is noted that the secondary peak appearing at the time of 21min is similar to the HPLC peak time of Brasenia schreberi polysaccharide, which indicates that the endophyte of Brasenia schreberi also has the ability to produce polysaccharide components similar to Brasenia schreberi polysaccharide besides producing specific polysaccharide peak.

Structural analysis of water shield polysaccharide and comparison of saccharide of endophyte

In order to further determine whether the Brasenia schreberi endophyte polysaccharide and the Brasenia schreberi polysaccharide also contain the same chemical functional group, infrared spectroscopy analysis was further performed in this example.

Partial strains are respectively picked from the bacteria in the LB culture medium plate and transferred to a purified strain to be inoculated in an LB liquid culture medium, and the strain is cultured for 2 days at the rotation speed of 200r/min and the temperature of 37 ℃ overnight under shaking. Centrifuging the bacterial liquid, removing protein from the supernatant by sevage method, performing alcohol precipitation with 4 times volume of ethanol, filtering, separating precipitate, and analyzing whether the precipitate or the alcohol precipitation liquid has glycosidic bond by infrared spectroscopy. The polysaccharide extracted from water shield is analyzed by infrared spectroscopy and compared with the pattern of precipitate or alcohol-separated liquid containing glycosidic bond.

The polysaccharide content of BSP is determined using the phenol-sulfuric acid method, using D-glucose as a standard. First, a control solution (0.1mg/mL) was prepared using D-glucose. Next, the reference solution was placed into tubes (0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL) and a phenol solution was added to allow it to stand. Concentrated sulfuric acid was then added rapidly and shaken. The tubes were left for 10min until they were cooled to room temperature and then the absorbance was measured at 490 nm. Redistilled water was used as a blank control. Results were determined using linear regression. The absorbance level of the sample is then measured to determine the uronic acid content.

The infrared spectroscopic analysis chart is shown in fig. 3, and fig. 3 shows that the water shield endophyte polysaccharide provided by the embodiment has 9 characteristic absorption peaks. As can be seen from the infrared spectrogram, 3392.79cm^-1The absorption peak at (B) is the stretching vibration absorption peak of-OH, 2924.09cm^-1Is a C-H stretching vibration absorption peak, which is a typical spectrum peak of two polysaccharides, and is similar to a typical spectrum peak of the water shield polysaccharide; 2854.65cm^-1、1558.48cm^-1And 1458.18cm^-The absorption peak is a C-H stretching vibration absorption peak on a benzene ring, and is a polysaccharide wave spectrum peak which is specific to the endosymbiotic bacteria of the water shield; 1653.00cm^-1The absorption peak is a carbon-oxygen double bond (C ═ O) absorption peak; 1404.18cm^-1，551.64cm^-1The absorption peaks are all the C-H stretching vibration absorption peaks. Of particular note, 1083.99cm^-1The absorption peak is with the glycosideThe absorption peak associated with the bond C-O-H is characteristic of β -glucan.

Compared with the water shield polysaccharide, the polysaccharide produced by the water shield endophyte is 3392.79cm^-1And 2924.09cm^-1Has the same polysaccharide absorption peak and a unique characteristic peak (at 2854.65 cm)^-1、1558.48cm^-1And 1458.18cm^-1). Mixing water shield endophytic bacteria and water shield polysaccharide at 1083.99cm^-1The characteristic peak of β -glucan is the same, the infrared spectrum analysis of the polysaccharide from different sources and the characteristic peak analysis are shown in the following table:

determination of polysaccharide antioxidant Activity

In this example, a hydroxyl radical kit provided by Nanjing as a built organism is used to detect a water shield polysaccharide extract. The polysaccharide sample and the reagent are mixed uniformly, react for 1 minute at 37 ℃, immediately add the color developing agent to stop the reaction, and are placed for 20 minutes at room temperature after being mixed uniformly. The spectrophotometer was zeroed with distilled water and the absorbance value of each tube was measured at 550 nm. The experimental result shows that the hydroxyl radical scavenging capacity is realized, and partial antioxidant activity of the water shield polysaccharide is obtained.

The importance of polysaccharides is attributed to their physiological activities. Hydroxy radical (A)^·OH) is a radical with strong oxidizing power, has active property, has a very fast oxidation rate of various organic and inorganic substances, is a main factor causing tissue lipid peroxidation, nucleic acid fragmentation, protein and polysaccharide decomposition, and is related to body aging, tumor, radiation damage and cell phagocytic power. In order to further determine the biological activity of the endosymbiont bacterial polysaccharide in water shield, the hydroxyl radical generating system in this example is used to compare the antioxidant activity of the endosymbiont bacterial polysaccharide in water shield. The results of the study showed that the inhibitory effect of the polysaccharides of the endosymbionts in water shield on the generation of hydroxyl radical was strongest at 1.0mg/mL (as shown in the following table), while the inhibitory effect of the polysaccharides of water shield on the generation of hydroxyl radical increased with the increase of the concentration (as shown in the following table). The experimental result shows that the brasenia schreberi endophyte polysaccharideThe cleaning effect on hydroxyl free radical is obviously higher than that of water shield polysaccharide.

Sequence listing

<110> university of the south China nationality

<120> a psychrobacter pneumococcus T-15 strain for producing polysaccharide and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1459

<212>DNA

<213> Acidophilus pneumophila (Psychrobacter pulmonis strain)

<400>1

gaatggcggg ccgctataat gctagtcgag cgaacagata aggagcttgc tcctttgacg 60

ttagcggcgg acgggtgagt aacacgtgga taacctacct ataagactgg gataacttcg 120

ggaaaccgga gctaataccg gataacatat tgaaccgcat ggttcaatag tgaaaggcgg 180

ctttgctgtc acttatagat ggatccgcgc cgtattagct agttggtaag gtaacggctt 240

accaaggcaa cgatacgtag ccgacctgag agggtgatcg gccacactgg aactgagaca 300

cggtccagac tcctacggga ggcagcagta gggaatcttc cgcaatgggc gaaagcctga 360

cggagcaacg ccgcgtgagt gatgaaggtc ttcggatcgt aaaactctgt tatcagggaa 420

gaacaaatgt gtaagtaact gtgcacatct tgacggtacc tgatcagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga attattgggc 540

gtaaagcgcg cgtaggcggt tttttaagtc tgatgtgaaa gcccacggct caaccgtgga 600

gggtcattgg aaactggaaa acttgagtgc agaagaggaa agtggaattc catgtgtagc 660

ggtgaaatgc gcagagatat ggaggaacac cagtggcgaa ggcgactttc tggtctgtaa 720

ctgacgctga tgtgcgaaag cgtggggatc aaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840

attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga attgacgggg 900

acccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccaaa 960

tcttgacatc ctttgaccgc tctagagata gagtcttccc cttcggggga caaagtgaca 1020

ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080

cgcaaccctt aagcttagtt gccatcatta agttgggcac tctaagttga ctgccggtga 1140

caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgat ttgggctaca 1200

cacgtgctac aatggacaat acaaagggca gctaaaccgc gaggtcaagc aaatcccata 1260

aagttgttct cagttcggat tgtagtctgc aactcgacta catgaagctg gaatcgctag 1320

taatcgtaga tcagcatgct acggtgaata cgttcccggg tcttgtacac accgcccgtc 1380

acaccacgag agtttgtaac acccgaagcc ggtggagtaa ccatttatgg agctagccgt 1440

cgagtggaga gctcagtct 1459

Claims (3)

1. A T-15 strain of Psychrobacter pneumococcus strain for producing polysaccharide is preserved in China center for type culture Collection with the preservation number: CCTCC M2019056, and the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.

2. Use of the T-15 strain of Achilles-pulmona as claimed in claim 1, characterized in that: used for preparing polysaccharide by fermentation.

3. The method for producing polysaccharides using the T-15 strain of Achilles-pulmona as described in claim 1, comprising the steps of: firstly, inoculating a psychrophilum apicomplexa T-15 strain into an LB liquid culture medium, carrying out shaking culture at the rotating speed of 200r/min and the temperature of 37 ℃ overnight for 2 days, centrifuging a zymogen solution at 12000r/min, and taking a supernatant; mixing the supernatant with chloroform-n-butanol solution in a centrifuge tube, oscillating for 20-30min, centrifuging, collecting supernatant, filtering the supernatant twice with 0.45 μm filter membrane, heating and concentrating at 70 deg.C to 1/2-2/3, adding 4 times volume of 95% ethanol, freezing in a refrigerator overnight, and centrifuging at 12000r/min for 10min to obtain precipitate as polysaccharide.

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