CN113322207B - Microbacterium XL1 and application thereof in Levan fructan production - Google Patents
Microbacterium XL1 and application thereof in Levan fructan production Download PDFInfo
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Abstract
The invention relates to the technical field of biological engineering, in particular to microbacterium XL1 and application thereof in Levan fructan production. The invention provides a preservation number of CCTCC NO: m2021423, and discloses the application of the strain in Levan fructan production, and the invention also provides the specific methods for strain screening and Levan fructan production culture. And through a plurality of tests, the optimal culture condition for the strain to produce the Levan fructan is provided, so that the utilization rate of a carbon source for producing the Levan fructan exceeds 64%.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to microbacterium XL1 and application thereof in Levan fructan production.
Background
The levan is a homopolysaccharide composed of fructofuranosyl connected by beta-2, 6 bonds and beta-2, 1 bonds, and is a biological macromolecule widely distributed in nature. Depending on the linkage, two types of fructans are distinguished: inulin and levan. Inulin, which is linked by β -2,1 glucosidic bonds, is usually derived from plants, and acts as a storage saccharide in the family Compositae and Poaceae, and thus inulin-type fructans can be isolated from natural plants. Levan is fructan composed of beta-2, 6 glycosidic linkages connecting D-fructofuranose residues as main chains and some beta-2, 1 glycosidic linkages as branch points, usually from microorganisms, and most of bacterial fructans are Levan-type fructans.
Levan type fructan has effects of anti-tumor, anti-diabetes, reducing blood lipid, enhancing immunity and increasing intestinal Bifidobacterium flora, and it also has heavy metal poisoning reducing effect.
Many levan fructan-producing microorganisms have been identified. Such as Bacillus circulans (Bacillus circulans), Bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus subtilis (Bacillus subtilis), Bacillus polymyxa (Bacillus polymyxa), Lactobacillus reuteri (Lactobacillus reuteri), Erwinia amylovora (Erwinia amylovora), Pseudomonas (Pseudomonas) and the like.
The yield of polysaccharide (i.e., the yield of levan fructan) in 200g/L sucrose medium by the strain Paenibacillus bovis BD3526 was 36.25 g/L. The levan yield of the pseudomonas menbergii is the highest and is 34.64g/L when the sucrose concentration of a culture medium is 150g/L, the yield is 41.7g/L when the sucrose concentration of the culture medium is 100g/L after the fermentation optimization of the polysaccharide from Bacillus licheniformis 8-37-0-1, and the utilization rate of a carbon source reaches 41.7%. It can be seen that the utilization rate of the carbon source for levan fructan produced by the existing strains still has a space to be improved.
Disclosure of Invention
The invention provides a microbacterium XL1 which can produce Levan fructan under mild conditions, and optimizes the conditions for producing Levan fructan, so that the utilization rate of a carbon source exceeds 64 percent, which is an unexpected effect.
The specific technical scheme is as follows:
a Microbacterium (Microbacterium) XL1, wherein the preservation number of the Microbacterium XL1 is CCTCC NO: m2021423.
Preferably, the growth medium of the microbacterium XL1 is MM solid medium, and the growth conditions are as follows: 25-45 ℃, and the MM solid culture medium is: 0.01g/L of ferrous sulfate heptahydrate, 0.1g/L of anhydrous calcium chloride, 1g/L of monopotassium phosphate, 0.15g/L of anhydrous magnesium chloride, 2.5g/L of sodium nitrate, 20g/L of cane sugar and 1.5 percent of agar, is prepared by pure water and has the pH value of 7.0-7.5. NaOH and HCl were used for pH adjustment of the medium.
The use of the aforementioned Microbacterium (Microbacterium) XL1 for the production of Levan fructan.
Preferably, the application method comprises the following steps:
and (3) culturing the microbacterium XL1 in a fermentation culture medium to obtain a fermentation liquid, wherein the fermentation liquid contains Levan fructan.
Further preferably, absolute ethyl alcohol is added into the fermentation liquor to precipitate polysaccharide.
Preferably, the microbacterium XL1 is firstly inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into a fermentation culture medium for culture.
Further preferably, the seed culture medium is: ferrous sulfate heptahydrate 0.01g/L, anhydrous calcium chloride 0.1g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium chloride 0.15g/L, NaNO30.05g/L, yeast extract 0.5g/L, tryptone 2.5g/L, sucrose 20g/L, prepared with pure water, pH 7.0-7.5.
Preferably, the culture conditions of the seed culture medium are as follows: the liquid loading is 20%, and the culture is carried out for 24h under the conditions of 30 ℃ and 180 rpm/min.
Preferably, the fermentation medium is: 0.01g/L of ferrous sulfate heptahydrate, 0.1g/L of anhydrous calcium chloride, 1g/L of potassium dihydrogen phosphate, 0.15g/L of anhydrous magnesium chloride, 1 g/L-5 g/L of nitrogen source and 70 g/L-90 g/L of cane sugar, wherein the pH value is 7.0-9.0, and the nitrogen source is selected from one or more of yeast extract, tryptone, urea, sodium nitrate and ammonium sulfate.
More preferably, the nitrogen source in the fermentation medium is 3g/L, and the sucrose is 70 g/L.
Preferably, the first and second liquid crystal materials are,the nitrogen sources in the fermentation medium are: 0.5g/L NaNO3And 2.5g/L yeast extract.
Preferably, the fermentation conditions of the fermentation medium are as follows: the temperature is 15-25 ℃, the fermentation time is 40-55 h, and the inoculation amount is 2-4%.
Advantageous effects
The invention provides a microbacterium XL1 which can be cultured under mild conditions to produce Levan fructan, and the whole fermentation process is simple to operate, the fermentation conditions are easy to control, and the microbacterium XL1 is suitable for industrialization. Furthermore, Levan fructan produced by the strain has high carbon source utilization rate.
Drawings
FIG. 1 is a photograph of Microbacterium XL1 cultured in MM solid medium for 48 hours;
FIG. 2 is a gram stain of Microbacterium XL 1;
FIG. 3 is a developmental tree of strain XL1 based on 16S rDNA sequence alignment of Microbacterium XL 1;
FIG. 4 is a graph showing the effect of carbon source species on extracellular polysaccharide production by Microbacterium XL 1;
FIG. 5 is a graph showing the effect of nitrogen source species on extracellular polysaccharide production by Microbacterium XL 1;
FIG. 6 is a graph showing the effect of carbon source content on extracellular polysaccharide production by Microbacterium XL 1;
FIG. 7 is a graph showing the effect of nitrogen source content on exopolysaccharide production by Microbacterium XL 1;
FIG. 8 is a graph showing the effect of different nitrogen source ratios on the extracellular polysaccharide production of Microbacterium XL 1;
FIG. 9 is a graph showing the effect of temperature on exopolysaccharide production by Microbacterium XL 1;
FIG. 10 is a graph showing the effect of pH on exopolysaccharide production by Microbacterium XL 1;
FIG. 11 is an elution curve of DEAE-Sepharose FF column chromatography exopolysaccharide;
FIG. 12 is an elution curve of purified exopolysaccharide from TOYOPEARL HW-50F column chromatography;
FIG. 13 is a diagram of the results of thin layer chromatography analysis of extracellular polysaccharide components XL1, which are glucose, arabinose, fructose, hydrolyzed extracellular polysaccharide and unhydrolyzed extracellular polysaccharide in the order from 1 to 6;
FIG. 14 is a high performance liquid chromatography analysis chart of extracellular polysaccharide component XL 1;
FIG. 15 is an FT-IR spectrum of XL1 exopolysaccharide;
FIG. 16 shows XL1 exopolysaccharide13A C-NMR spectrum;
FIG. 17 shows XL1 exopolysaccharide1H-NMR spectrum;
FIG. 18 is a 2D-HSQC spectrum of XL1 exopolysaccharide;
FIG. 19 shows the 2D-HMBC spectrum of XL1 exopolysaccharide.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention will be described in detail below with reference to the attached drawings to facilitate understanding of the present invention by those skilled in the art.
The inoculum size referred to in the present invention refers to the volume of the seed solution transferred and the ratio of the culture solution before inoculation.
The yeast extract in the present invention refers to a yeast extract, which may be in the form of powder or paste.
The yeast extracts used in the examples of the present invention were: OXOID LP 0021.
The strain Microbacterium XL1 related to the invention is a bacterium (Microbacterium sp.XL1) separated from wetland soil of Bay of continuous cloud harbor, the strain is preserved in China center for type culture Collection at 21/4/2021, the culture viability is detected by the collection center at 28/4/2021, the detection result is survival, and the collection number is: CCTCC NO: m2021423, depository address: wuhan, Wuhan university, contact phone: (027)68754052.
1. Screening method of the Strain of the present invention
(1) Preparation of the culture medium used in the embodiment of the present invention:
unless otherwise specified, the following formulations were used for each of the media used in the following examples:
MM solid medium: 0.01g/L of ferrous sulfate heptahydrate, 0.1g/L of anhydrous calcium chloride, 1g/L of monopotassium phosphate, 0.15g/L of anhydrous magnesium chloride, 2.5g/L of sodium nitrate, 20g/L of sucrose and 1.5 percent of agar, preparing with pure water, and adjusting the pH value to 7.0-7.5 by using 5M NaOH and HCl;
seed culture medium: 0.01g/L ferrous sulfate heptahydrate, 0.1g/L anhydrous calcium chloride, 1g/L potassium dihydrogen phosphate, 0.15g/L anhydrous magnesium chloride, 0.05g/L sodium nitrate, 0.5g/L yeast extract, 2.5g/L tryptone and 20g/L sucrose, preparing with pure water, and adjusting pH to 7.0-7.5 with 5M NaOH and HCl;
fermentation medium: 0.01g/L ferrous sulfate heptahydrate, 0.1g/L anhydrous calcium chloride, 1g/L potassium dihydrogen phosphate, 0.15g/L anhydrous magnesium chloride, 0.5g/L sodium nitrate, 2.5g/L yeast extract and 70g/L sucrose, wherein the pH value is adjusted to 7.0-7.5 by using 5M NaOH and HCl.
(2) The screening method of the strain comprises the following steps:
adding 50ml of normal saline into 1g of soil sample to prepare bacterial suspension, sucking 200 mul of bacterial suspension on MM solid culture medium under aseptic condition for coating and inoculation, culturing at 30 ℃ for 36h, selecting bacterial colony suspected of producing sugar on MM solid culture medium, diluting with normal saline, selecting 10-3、10-5、10-7Diluting and coating the bacterial liquid with the concentration, and culturing at the constant temperature of 30 ℃ for 36 hours again; and selecting a single colony for dilution and coating, repeatedly carrying out operation until a purified strain is obtained, and screening a sugar-producing strain required by an experiment, namely the microbacterium XL 1.
2. The strain XL1 has morphological characteristics and physiological and biochemical characteristics.
2.1 morphological characteristics:
the strain is gram-positive bacteria, the gram staining of the strain is purple rod-shaped, and the result is shown in figure 2.
After 48h of culture in MM solid medium, the colonies were round and transparent as shown in FIG. 1.
2.2 physiological and biochemical characteristics:
the strain can utilize mannitol, arabinose, sorbitol and bitter apricot kernel, and the physiological and biochemical results are shown in table 1:
TABLE 1 physio-biochemical characteristics of Strain XL1
In table 1, +: positive; -: and (4) negativity.
2.3 molecular biological characterization of Strain XL1
Extracting the genome of the strain XL1 by using a bacterial genome extraction kit, and selecting a universal primer for amplifying a prokaryotic microorganism 16SrDNA sequence: (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3') in a 50. mu.l reaction system: DDwater 39.5. mu.l, PCR buffer 5. mu.l, Mg 2+1. mu.l of each of the upstream and downstream primers, 1. mu.l of dNTP, 1. mu.l of template DNA, 0.5. mu.l of Taq plus ploymerase, reaction conditions: denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 2min15s, 30 cycles, and 5min at 72 deg.C; agarose electrophoresis of PCR products is purified and recovered, a cloning vector is constructed, positive clones are selected and sent to Shanghai engineering for sequencing, the determined sequences are reversely and complementarily spliced to obtain a sequence, the 16SrDNA sequence of the strain is shown as SEQID NO 1, the strain can be preliminarily determined to be Microbacterium (Microbacterium) through homology comparison of the 16SrDNA sequence, the strains with the closer genetic relationship are subjected to multiple comparison by MEGA software, a phylogenetic tree is built by using a middle abutment-joining method, and the relationship between the strain XL1 and the Microbacterium is shown to be nearest from the phylogenetic tree (see figure 3).
SEQID NO.1：
TGGTGTGGCGGCAGCTTACCATGCAAGTCGTACGGTGTAGCCAAGCTTGCTTGGTGGATCAGTGGCGAACGGGTGAGTAACACGTGAGCAACCTGCCCTGGACTCTGGGATAAGCGCTGGAAACGGTGTCTAATACTGGATATGAGGCGTGATCGCATGGTCATGTTTGGAAAGATTTTTCGGTCTGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGTGAGTGACGGTACCTGCAGAAAAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCCGAGGCTCAACCTCGGGCCTGCAGTGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGGAGCAAACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGACCATTCCACGGTTTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCAGAACACCGTAGAAATACGGGACTCTTTGGACACTGGTGAACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCAATACCGTGAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAAGTCATGAAAGTCGGTAACACCTGAAGCCGGTAGCAGTAACATCTTGTGGACGGAGCCGTCGAAGGTGCATTCTGTTCC
3. Optimization of sugar production fermentation of strain XL1
3.1 preparation of seed solutions
Inoculating the strain XL1 into an MM solid culture medium, culturing for 2d in an incubator at 30 ℃, inoculating a single colony on the culture medium into a seed culture medium, filling the liquid to 20%, and culturing for 24h at 30 ℃ and 180rpm/min to obtain a seed liquid for later use.
3.2 Strain XL1 fermentation for sugar production
(1) Inoculating the seed solution into a fermentation culture medium, wherein the inoculation amount is 2-4%, and performing shaking culture at the constant temperature of 20 ℃ for 48 hours to obtain fermentation liquor.
(2) Adding 1-2 times volume of anhydrous ethanol into the fermentation broth to separate out polysaccharide, standing for 1-2h, centrifuging to obtain precipitate, and oven drying to obtain crude polysaccharide; re-dissolving the crude polysaccharide, removing protein by Sevag method for 3-5 times, precipitating polysaccharide with 1-2 times volume of anhydrous ethanol, and centrifuging at 8000rpm/min for 15min to obtain precipitate as crude polysaccharide; mixing and dissolving a proper amount of crude polysaccharide and deionized water, dialyzing in a dialysis bag overnight, replacing the deionized water every 4h midway, and freeze-drying in vacuum; dissolving the dialyzed polysaccharide with deionized water, and further purifying by anion exchange chromatography column and gel column chromatography to obtain Microbacterium XL1 extracellular polysaccharide.
3.3 fermentation Condition optimization
The following references to the content of each substance in the medium mean the per g/100ml meaning, for example, the following 5% carbon source means that the content of the carbon source is 5g/100ml, that is, 50 g/L; the nitrogen source of 0.3% described below means that the content of the nitrogen source is 0.3g/100mL, that is, 3 g/L.
(1) Influence of carbon source on yield of extracellular polysaccharide of Microbacterium XL1
The fermentation medium comprises the following components: ferrous sulfate heptahydrate 0.01g/L, anhydrous calcium chloride 0.1g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium chloride 0.15g/L, NaNO30.05g/L, yeast extract 0.5g/L, tryptone 2.5g/L, sucrose 50g/L, prepared in purified water, adjusted to pH7.0 with 5M NaOH and HCl.
The method comprises the steps of replacing sucrose in the culture medium with 5% of different carbon sources (sucrose, glucose, fructose and maltose), culturing for 48 hours under the conditions of 20 ℃ and 180rpm/min according to the inoculation amount of 4% without changing other components, precipitating fermentation liquor by 2 times of volume of absolute ethyl alcohol, centrifuging at 8000rpm/min for 15min to obtain crude polysaccharide, directly drying, weighing, and obtaining a test result shown in figure 4.
(2) Influence of nitrogen source on yield of extracellular polysaccharide of Microbacterium XL1
The fermentation medium comprises the following components: ferrous sulfate heptahydrate 0.01g/L, anhydrous calcium chloride 0.1g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium chloride 0.15g/L, NaNO30.05g/L, yeast extract 0.5g/L, tryptone 2.5g/L, sucrose 50g/L, prepared in purified water, adjusted to pH7.0 with 5M NaOH and HCl.
With 0.3% of different nitrogen sources (yeast extract, tryptone, urea)Sodium nitrate, ammonium sulfate) in place of NaNO in the above medium, respectively3The yeast extract and the nitrogen source represented by the tryptone are cultured for 48 hours at 20 ℃ under the condition of 180rpm/min according to the inoculation amount of 4 percent, 2 times of volume of absolute ethyl alcohol is used for precipitating the fermentation liquor, the fermentation liquor is centrifuged at 8000rpm/min for 15min to obtain crude polysaccharide through precipitation, and the crude polysaccharide is dried and weighed, and the test result is shown in figure 5, and the result shows that the yeast extract is more favorable for producing sugar by Microbacterium XL1, but the polysaccharide producing capacity of the tryptone, urea, sodium nitrate and ammonium sulfate is not weak.
(3) Influence of carbon source content on yield of extracellular polysaccharide of Microbacterium XL1
The culture medium comprises the following components: ferrous sulfate heptahydrate 0.01g/L, anhydrous calcium chloride 0.1g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium chloride 0.15g/L, NaNO30.05g/L, yeast extract 0.5g/L, tryptone 2.5g/L, sucrose 50g/L, prepared in purified water, adjusted to pH7.0 with 5M NaOH and HCl.
The sucrose content in the culture medium is replaced by different sucrose contents (5%, 7%, 10%, 15%, 20% and 25%), other components are unchanged, the culture is carried out for 48 hours under the conditions of 20 ℃ and 180rpm/min according to the inoculation amount of 4%, 2 times of volume of absolute ethyl alcohol is used for precipitating fermentation liquor, the fermentation liquor is centrifuged at 8000rpm/min for 15min to obtain crude polysaccharide through precipitation, the crude polysaccharide is dried and weighed, the test result is shown in figure 6, the result shows that the sugar yield of Microbacterium XL1 is increased along with the increase of the sucrose content, when the sucrose content is 20%, the sugar yield is 73g/L and reaches the highest, the yield is not increased even when the sucrose is continuously increased, and when the sucrose content is 7%, the utilization rate of a carbon source is 58.9% at the highest.
(4) Influence of nitrogen source content on yield of extracellular polysaccharide of Microbacterium XL1
The culture medium comprises the following components: 0.01g/L ferrous sulfate heptahydrate, 0.1g/L anhydrous calcium chloride, 1g/L potassium dihydrogen phosphate, 0.15g/L anhydrous magnesium chloride, 3g/L yeast extract and 70g/L sucrose, prepared with pure water, and adjusted to pH7.0 with 5M NaOH and HCl.
Replacing the yeast extract content of the culture medium with different yeast extract contents (0.1%, 0.2%, 0.3%, 0.4%, 0.5%), keeping the other components unchanged, culturing for 48h at 20 ℃ and 180rpm/min according to the inoculation amount of 4%, precipitating the fermentation liquor by using 2 times of volume of absolute ethyl alcohol, centrifuging at 8000rpm/min for 15min to obtain crude polysaccharide, drying and weighing, wherein the test result is shown in fig. 7, and the result shows that when the yeast extract content is 0.3%, the yeast extract content is most beneficial to the sugar production of the microbacterium XL 1.
(5) Influence of different nitrogen source ratios on yield of extracellular polysaccharide of Microbacterium XL1
The composition of the culture medium is as follows: ferrous sulfate heptahydrate 0.01g/L, anhydrous calcium chloride 0.1g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium chloride 0.15g/L, NaNO30.5g/L, yeast extract 2.5g/L, sucrose 70g/L, prepared in purified water, adjusted to pH7.0 with 5M NaOH and HCl.
Different nitrogen source ratios (0.3% yeast extract, 0.25% yeast extract + 0.05% sodium nitrate, 0.15% yeast extract + 0.15% sodium nitrate, 0.05% yeast extract + 0.25% sodium nitrate) were used instead of yeast extract and NaNO in the medium3The proportion and other components are unchanged, the fermentation liquor is cultured for 48 hours at 20 ℃ and 180rpm/min according to the inoculation amount of 4 percent, ethanol is used for precipitating the fermentation liquor to obtain crude polysaccharide, the crude polysaccharide is dried and weighed, and the result is shown in figure 8, when the proportion of the yeast extract and the sodium nitrate is 5: 1, the yeast extract is most beneficial to the sugar production of the microbacterium XL1, and the utilization rate of the carbon source exceeds 64 percent.
(5) Influence of temperature on yield of extracellular polysaccharide of Microbacterium XL1
The culture medium comprises the following components: ferrous sulfate heptahydrate 0.01g/L, anhydrous calcium chloride 0.1g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium chloride 0.15g/L, NaNO30.5g/L, yeast extract 2.5g/L, sucrose 70g/L, prepared in purified water, adjusted to pH7.0 with 5M NaOH and HCl.
Preparing a fermentation liquid culture medium according to the composition of the culture medium, inoculating seed liquid into the fermentation culture medium with the inoculation amount of 4%, rotating at the speed of 180r/min, respectively culturing at different temperatures (10 ℃, 15 ℃, 20 ℃, 25 ℃ and 30 ℃) for 48h, precipitating the fermentation liquid by using 2 times of volume of absolute ethyl alcohol, centrifuging at 8000rpm/min for 15min to obtain crude polysaccharide through precipitation, drying and weighing, wherein the test result is shown in figure 9, the test result shows that the fermentation temperature is 15-25 ℃ and is more favorable for producing sugar by Microbacterium XL1, the preferable temperature is 15-20 ℃, and the optimal temperature is 20 ℃.
(6) Effect of pH on extracellular polysaccharide production of Microbacterium XL1
The culture medium comprises the following components: ferrous sulfate heptahydrate 0.01g/L, anhydrous calcium chloride 0.1g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium chloride 0.15g/L, NaNO30.5g/L, yeast extract 2.5g/L, sucrose 70g/L, prepared with purified water, adjusted to pH7.0-7.5 with 5M NaOH and HCl.
The method comprises the steps of configuring a fermentation culture medium according to the culture medium, inoculating seed liquid into the fermentation culture medium which is prepared by 5M NaOH and HCl solutions and has different pH values (6, 7, 8, 9 and 10) at an inoculation amount of 4%, culturing for 48 hours at a rotation speed of 180rpm/min, precipitating fermentation liquor by using 2 times of volume of absolute ethyl alcohol, centrifuging at 8000rpm/min for 15min to obtain precipitate to obtain crude polysaccharide, drying and weighing, wherein the test result is shown in figure 10, the test result shows that the method is more favorable for producing sugar by microbacterium XL1 when the pH value is 7-9, and the optimal pH value is 7.
4. Extraction of strain XL1 exopolysaccharide
Dissolving the crude polysaccharide obtained by the method in deionized water to re-dissolve into 5% sugar solution, mixing with Sevag solution (chloroform: n-butyl alcohol is 4: 1) in equal volume, shaking the mixed solution vigorously for 10min, centrifuging at 8000rpm/min for 10min, taking supernatant, repeating the steps to mix the supernatant with Sevag solution, shaking and centrifuging for 4-6 times, precipitating the finally obtained supernatant with 2 times volume of absolute ethyl alcohol, centrifuging at 8000rpm/min for 15min to take precipitate, drying, re-dissolving 0.1g of dried polysaccharide in 10ml of deionized water, measuring sugar content by phenol-sulfuric acid method and protein content by BCA protein measuring kit, and measuring protein content by y 2.6276x +0.1201(R is 4: 1)20.9969) is the protein standard equation where x is the absorbance value at 562nm and y is the protein content. The results show that the protein content is almost negligible.
5. Column chromatography for purifying polysaccharide
(1) DEAE-Sepharose FF column chromatography
Dissolving the crude polysaccharide after deproteinization by the method of section 4 in deionized water to form a sugar solution with a certain concentration, subjecting DEAE-Sepharose FF column (4X 20cm) to equilibration for 2-3 column volumes with 10mM Tris-HCl (pH7.2) solution, slowly adding the sugar solution to the equilibrated column, then eluting with 10mM Tris-HCl (pH7.2) solution and 10mM Tris-HCl (pH7.2) solution with different NaCl concentration, respectively, eluting 2 column volumes for each gradient, with a flow rate of 2mL/min, collecting with a distribution collector, measuring sugar content by phenol-sulfuric acid method according to the number of tubes, collecting the main part of polysaccharide peak, dialyzing, vacuum freeze-concentrating, and finding out the result as shown in FIG. 11, showing that the extracellular polysaccharide elutes one peak each at 10mM Tris-HCl (pH7.2) solution and 10mM Tris-HCl (pH7.2) solution with 0.1M NaCl concentration, and selecting the main peak to collect the peak eluted with 10mM Tris-HCl (pH7.2) solution.
(2) TOYOPEARL HW-50F column chromatography
Dissolving polysaccharide obtained by DEAE-Sepharose FF column chromatography, adding into TOYOPEARL HW-50F chromatographic column (2.6 × 70cm) with 2-3 column volumes balanced with deionized water, eluting with deionized water at flow rate of 0.4mL/min, collecting eluted components with distribution collector, eluting 2-3 column volumes, measuring sugar content by phenol-sulfuric acid method according to tube number, vacuum freeze drying main part of collected peak, and showing only one peak in figure 12, which shows that the polysaccharide has uniform molecular weight.
6. Extracellular polysaccharide composition analysis
(1) Thin layer chromatography
Acid hydrolysis of extracellular polysaccharide: weighing 10mg of extracellular polysaccharide finally purified in section 5, dissolving the extracellular polysaccharide with a proper amount of deionized water, placing the solution in a test tube with a plug, slowly dropwise adding sulfuric acid, uniformly mixing until the concentration reaches 0.5M, hydrolyzing at 80 ℃ for 3h to completely hydrolyze the extracellular polysaccharide, adding barium carbonate into hydrolysate to neutralize the hydrolysate until no bubbles exist, and centrifuging to obtain supernatant as hydrolyzed extracellular polysaccharide. 2% monosaccharide standard (glucose, arabinose and fructose) solution, hydrolyzed fructose, hydrolyzed extracellular polysaccharide and unhydrolyzed extracellular polysaccharide are sequentially spotted on a TLC plate, then the TLC plate is placed in a spreading agent (n-butanol: acetone: water is 4: 3: 1) for spreading, diphenylamine-aniline-phosphoric acid (DAP) color developing agent is used for developing, then the TLC plate is placed in an oven at 80 ℃ for standing for 10min, and the observation result is shown in figure 13, wherein the serial numbers 1 to 6 are glucose, arabinose, fructose, hydrolyzed extracellular polysaccharide and unhydrolyzed extracellular polysaccharide in sequence, and the XL1 extracellular polysaccharide is preliminarily judged to be composed of fructose.
(2) High performance liquid chromatography
Preparing 10mg/ml monosaccharide standard (glucose, fructose, galactose) and hydrolyzed exopolysaccharide, filtering with 0.22 μm filter, and performing high performance liquid detection, wherein Sugar-Pak I chromatographic column is used, the flow rate is 0.4ml/min, the detector is a differential refraction detector, the mobile phase is deionized water, the column temperature is 80 deg.C, and the sample volume is 20 μ l. The test result is shown in figure 13, the upper part of figure 13 is a detection peak of a monosaccharide standard, the peaks are detection peaks of glucose, galactose and fructose in sequence from left to right, the retention time of the glucose, the galactose, the fructose and the hydrolyzed extracellular polysaccharide is respectively 12.4, 13.7, 15.2 and 15.2min, the lower part of figure 14 is a detection peak of the hydrolyzed extracellular polysaccharide, the peak-off time is 15.2min, the retention time of the hydrolyzed extracellular polysaccharide is consistent with the fructose as shown in the figure, the XL1 extracellular polysaccharide is shown to be composed of the fructose, and the high performance liquid phase result is consistent with the thin layer chromatography.
7. Extracellular polysaccharide structural analysis
(1) Fourier Infrared Spectroscopy
Mixing and grinding 5mg of purified polysaccharide powder and 100mg of potassium bromide, drying in an oven to constant weight, tabletting, and placing on an infrared spectrometer at 4000-400 cm-1The wavelength range is scanned. The results are shown in FIG. 15, which shows 925cm in an infrared spectral scan-1And 809cm-1The characteristic absorption peak at (a) indicates that the polysaccharide contains furan rings.
(2) Nuclear magnetic resonance analysis
Preparing 120mg/ml purified polysaccharide solution, hydrolyzing in 0.01M sulfuric acid solution at 60 deg.C for 3min until sugar solution has no viscous feeling, neutralizing with barium carbonate until no bubble is generated, centrifuging to obtain supernatant, dialyzing with deionized water, vacuum freeze drying, adding lyophilized polysaccharide powder into D2Lyophilize again in O, repeat three times, and take 30mg of the treated sample in 500. mu.L of water [ 99.9% D2O and 0.05% (w/v) (trimethylsilyl) sodium deuteropropionate (TSP)]Then, the mixture was centrifuged at 12000rpm/min for 10min, and the supernatant was added to a clean nuclear magnetic tube. NMR analysis Using a Bruker AV-500(500MHz) NMR spectrometer, the measurement temperature was 30 ℃ and the measurement was carried out13CNMR、1HNMR, 2D-HSQC and 2D-HMBC spectrograms show that the results are shown in FIGS. 16-19, and XL1 exopolysaccharide is beta- (2,6) -D-Fructan, namely levan type Fructan through spectrogram analysis.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
SEQUENCE LISTING
<110> university of oceanic Jiangsu
<120> Microbacterium XL1 and application thereof in Levan fructan production
<130> 20210607
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1433
<212> DNA
<213> Artificial Synthesis
<400> 1
tggtgtggcg gcagcttacc atgcaagtcg tacggtgtag ccaagcttgc ttggtggatc 60
agtggcgaac gggtgagtaa cacgtgagca acctgccctg gactctggga taagcgctgg 120
aaacggtgtc taatactgga tatgaggcgt gatcgcatgg tcatgtttgg aaagattttt 180
cggtctggga tgggctcgcg gcctatcagc ttgttggtga ggtaatggct caccaaggcg 240
tcgacgggta gccggcctga gagggtgacc ggccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgcaagcctg atgcagcaac 360
gccgcgtgag ggatgacggc cttcgggttg taaacctctt ttagcaggga agaagcgtga 420
gtgacggtac ctgcagaaaa agcgccggct aactacgtgc cagcagccgc ggtaatacgt 480
agggcgcaag cgttatccgg aattattggg cgtaaagagc tcgtaggcgg tttgtcgcgt 540
ctgctgtgaa atcccgaggc tcaacctcgg gcctgcagtg ggtacgggca gactagagtg 600
cggtagggga gattggaatt cctggtgtag cggtggaatg cgcagatatc aggaggaaca 660
ccgatggcga aggcagatct ctgggccgta actgacgctg aggagcgaaa gggtggggag 720
caaacaggct tagataccct ggtagtccac cccgtaaacg ttgggaacta gttgtgggga 780
ccattccacg gtttccgtga cgcagctaac gcattaagtt ccccgcctgg ggagtacggc 840
cgcaaggcta aaactccaaa ggaattgacg gggacccgca caagcggcgg agcatgcgga 900
ttaattcgat gcaacgcgaa gaaccttacc aaggcttgac atacaccaga acaccgtaga 960
aatacgggac tctttggaca ctggtgaaca ggtggtgcat ggttgtcgtc agctcgtgtc 1020
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctc gttctatgtt gccagcacgt 1080
aatggtggga actcatggga tactgccggg gtcaactcgg aggaaggtgg ggatgacgtc 1140
aaatcatcat gccccttatg tcttgggctt cacgcatgct acaatggccg gtacaaaggg 1200
ctgcaatacc gtgaggtgga gcgaatccca aaaagccggt cccagttcgg attgaggtct 1260
gcaactcgac ctcatgaagt cggagtcgct agtaatcgca gatcagcaac gctgcggtga 1320
atacgttccc gggtcttgta cacaccgccc gtcaagtcat gaaagtcggt aacacctgaa 1380
gccggtagca gtaacatctt gtggacggag ccgtcgaagg tgcattctgt tcc 1433
Claims (12)
1. Microbacterium (Microbacterium) XL1, characterized in that the Microbacterium XL1 has the collection number CCTCC NO: m2021423.
2. Microbacterium (Microbacterium) XL1 according to claim 1, wherein the growth medium of the Microbacterium XL1 is MM solid medium and the growth conditions are: 25-45 ℃, and the MM solid culture medium is: 0.01g/L of ferrous sulfate heptahydrate, 0.1g/L of anhydrous calcium chloride, 1g/L of monopotassium phosphate, 0.15g/L of anhydrous magnesium chloride, 2.5g/L of sodium nitrate, 20g/L of cane sugar and 1.5 percent of agar, is prepared by pure water and has the pH value of 7.0-7.5.
3. Use of the Microbacterium (Microbacterium) XL1 according to claim 1 for the production of Levan fructan.
4. The application according to claim 3, wherein the method of application is:
the microbacterium XL1 according to claim 1 is cultured in a fermentation medium to obtain a fermentation broth, i.e., a broth containing Levan fructan.
5. The use according to claim 4, wherein anhydrous ethanol is added to the fermentation broth to precipitate polysaccharides.
6. The use of claim 4, wherein the microbacterium XL1 of claim 1 is inoculated into a seed culture medium to be cultured to obtain a seed solution, and then the seed solution is inoculated into a fermentation culture medium to be cultured.
7. Use according to claim 6, wherein the seed culture medium is: ferrous sulfate heptahydrate 0.01g/L, anhydrous calcium chloride 0.1g/L, potassium dihydrogen phosphate 1g/L, anhydrous magnesium chloride 0.15g/L, NaNO30.05g/L, yeast extract 0.5g/L, tryptone 2.5g/L, sucrose 20g/L, prepared with pure water, pH 7.0-7.5.
8. Use according to any one of claims 6 to 7, wherein the seed culture medium is cultured under conditions selected from the group consisting of: the liquid loading is 20%, and the culture is carried out for 24h under the conditions of 30 ℃ and 180 rpm/min.
9. Use according to any one of claims 4 to 7, wherein the fermentation medium is: 0.01g/L of ferrous sulfate heptahydrate, 0.1g/L of anhydrous calcium chloride, 1g/L of potassium dihydrogen phosphate, 0.15g/L of anhydrous magnesium chloride, 1 g/L-5 g/L of nitrogen source and 70 g/L-90 g/L of cane sugar, wherein the pH value is 7.0-9.0, and the nitrogen source is selected from one or more of yeast extract, tryptone, urea, sodium nitrate and ammonium sulfate.
10. The use according to claim 9, wherein the fermentation medium contains 3g/L of nitrogen source and 70g/L of sucrose.
11. The use according to claim 10, wherein the nitrogen source in the fermentation medium is: 0.5g/LNaNO3And 2.5g/L yeast extract.
12. Use according to any one of claims 4 to 7, wherein the fermentation conditions of the fermentation medium are: the temperature is 15-25 ℃, the fermentation time is 40-55 h, and the inoculation amount is 2-4%.
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