CN108239613A - A kind of feed series bacillus, its culture medium and the application in sea grass polysaccharide degrading enzyme is prepared - Google Patents
A kind of feed series bacillus, its culture medium and the application in sea grass polysaccharide degrading enzyme is prepared Download PDFInfo
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Abstract
The present invention relates to a kind of microorganisms technical field more particularly to feed series bacillus, its culture medium and applications in sea grass polysaccharide degrading enzyme is prepared.The present invention provides the feed series bacillus that deposit number is CGMCC NO.12912, and the present invention provides the culture medium for screening strain, the culture medium for activated spawn and the culture mediums for fermentation.And provide with the strain fermentation prepare sea grass polysaccharide degrading enzyme method and fermentation made from sea grass polysaccharide degrading enzyme.There is good enzymatic activity with the sea grass polysaccharide degrading enzyme that strain provided by the invention and fermentation generate, can degrade sea grass polysaccharide, and the sea grass polysaccharide oligosaccharides of different polymerization degree can be prepared according to different parameters.Experiment shows the activity of sea grass polysaccharide degrading enzyme provided by the invention up to 1.03U/mL.
Description
Technical field
The present invention relates to microorganisms technical field more particularly to a kind of feed series bacillus, its culture medium and preparing
Application in sea grass polysaccharide degrading enzyme.
Background technology
Seaweed polysaccharide sulfate is the sulfate heteroglycan extracted from seaweed, structure sulfur-bearing acid groups, monosaccharide group
Into having glucose (Glc), rhamnose (Rha), xylose (Xyl), glucuronic acid (GlcA), mannose (Man) and galactolipin
(Gal) etc., sulfate radical is mainly connected on rhamnose, but the difference due to difference of Enteromorpha type and extracting method.
Existing research confirms that sea grass polysaccharide has immunological regulation, antitumor, anticoagulation, antiviral, anti-oxidant, reducing blood lipid
Etc. a variety of physiological active functions.Also, sea grass polysaccharide is because with excellent heat-convertible gel, the hydrophilic performances such as nontoxic.Therefore,
Sea grass polysaccharide and be applied to food, drug, cosmetics or chemical field potentiality.As people are to sea grass polysaccharide structure and work(
The deep understanding of energy, application field can also be widened constantly.
But since sea grass polysaccharide relative molecular mass is excessive, its dissolubility and absorbability is made to be affected, limit its
Application in field of medicaments and cosmetics.For sea grass polysaccharide oligosaccharides compared with sea grass polysaccharide, relative molecular mass is smaller, dissolubility,
Stability and safety all increased, and bioactivity also has and is improved to some extent.
The method that degradation sea grass polysaccharide prepares sea grass polysaccharide oligosaccharides at present has Physical, chemical method.Acid drop in chemical method
Solution is the conventional method of polysaccharide hydrolysis, and the hydrolysis that different palliating degradation degrees can be obtained by adjusting acidity, temperature and action time is produced
Object.But sour water solution action condition is violent, poor to Substratspezifitaet, and in degradation process sulfated polysaccharide sulfate easily by water
Solution comes off, and the reduction of sulfate content can cause the physiological activity of polysaccharide to reduce.Though oxidative degradation can reduce polysaccharide in degradation process
In sulfate come off, but oxidation product is relatively complicated, increase the difficulty isolated and purified.
Enzyme process, which prepares oligosaccharides, has specificity, and optionally the specific site on enzymolysis cut-out sugar chain, can also avoid sulphur
Acidic group comes off, so as to which specific oligosaccharides be made；And the method reaction condition is mild, and degradation process is easy to control, also with cost
Cheap, simple for process, many advantages, such as yield is high, pollution-free.But the high enzyme of hydrolysis efficiency is difficult to obtain, at present to sea grass polysaccharide
Enzymic degradation seldom have been reported.Therefore, the microorganism of sea grass polysaccharide can be utilized by filtering out, and therefrom extract sea grass polysaccharide
Degrading enzyme is used to prepare sea grass polysaccharide low molecular weight active fragment, becomes the important directions of the high-valued research of sea grass polysaccharide industry,
There is wide development potentiality in terms of the exploitation of marine drug simultaneously.
Invention content
In view of this, the technical problem to be solved in the present invention be to provide a kind of feed series bacillus, its culture medium and
Application in sea grass polysaccharide degrading enzyme is prepared, feed bacillus provided by the invention can generate sea grass polysaccharide degrading enzyme,
The enzyme activity of its sea grass polysaccharide degrading enzyme generated is up to 1.03U/mL.
The bacterial strain for generating sea grass polysaccharide degrading enzyme is obtained in order to screen, the present invention provides screening and culturing medium, wherein
Component including water and following mass fraction：
The pH value of the culture medium is 7.0.
The MgSO4Hydrate be MgSO4·7H2O；The CaCl2Hydrate be CaCl2·7H2O；The FePO4
Hydrate be FePO4·7H2O。
In some specific embodiments, screening and culturing medium includes the component of water and following mass fraction：
Agar can also be added in the screening and culturing medium, solid medium is made.
In solid medium, the mass fraction of agar is 1%~5%.Preferably, the mass fraction of agar is 2%.
Application of the screening and culturing medium provided by the invention in screening can generate the bacterial strain of sea grass polysaccharide degrading enzyme.
The screening technique that the bacterial strain of sea grass polysaccharide degrading enzyme can be generated includes：
Step 1：Ooze with antiseptic sea water is dissolved, is seeded to screening and culturing medium provided by the invention, shaking flask culture 2~3
My god；
Step 2：Culture solution in step 1 is coated on solid screening and culturing medium culture 3~5 days；
Step 3：The solid screening and culturing medium through culture in step 2 is dyed with Lugol's iodine solution, degradation will be generated
The bacterium colony of circle, which is crossed to new solid screening and culturing medium, to be continued to cultivate；
Step 4：Step 3 is repeated, until obtaining single culture.
The ooze that the present invention uses comes from Qingdao of Shandong province No.One Bathing Beach surrounding waters.
The temperature of step 1 shaking flask culture is 28 DEG C, rotating speed 200rpm.
The temperature that step 2 is cultivated is 28 DEG C.
After being screened by the method provided by the present invention, the bacterium colony picking for generating most degradation circle is subjected to biological deposits.Its
Deposit number is CGMCC NO.12912.
The present invention provides the feed series bacillus that deposit number is CGMCC NO.12912.
The biochemical characteristics of the bacterial strain includes：
Colony morphology characteristic：Good in the cultured on solid medium containing sea grass polysaccharide, bacterium colony milky is round, table
Face is slightly raised, smooth, non-reflective, opaque, neat in edge, and diameter is about 3.0mm；
Morphological features：Cell is in direct rod shape, 0.6~0.8 1.8~3.5 μm of μ m, single arrangement, Gram's staining
The positive, uniform coloring can produce pod membrane, move (peritrichous)；It is raw or near middle raw in gemma, it is wide less than or equal to cell, in circle
Shape or ellipse.
Physiological and biochemical property：Sea grass polysaccharide degrading enzyme can be generated in sea grass polysaccharide fluid nutrient medium；It is aerobic；It can utilize
Alpha-cyclodextrin, beta-cyclodextrin, dextrin, starch, amarogentin, ursin, cellobiose, D-Fructose, D- galactolipins, rough gentian two
Sugar, alpha-D-glucose, α-D- lactose, lactose, maltose, mannose, PEARLITOL 25C, D-MANNOSE, melezitose, melibiose, α-
Methyl D-galactoside, Beta-methyl-D- galactosides, 3- methyl-glucose, Alpha-Methyl-D-Glucose glycosides, Beta-methyl-D- Portugals
Polyglycoside, Alpha-Methyl-D-MANNOSE glycosides, isomaltoketose, D-Psicose, D- raffinoses, salicin, D-glucitol, wood
Sugar, sucrose, D-Tag, D- trehaloses, turanose, acetic acid, beta-hydroxybutyric acid, methyl pyruvate, pyruvic acid, succinic acid list first
Ester, glycerine, adenosine, 2'- desoxyadenossines, inosine, thymidine, uridine, adenosine 5'-phosphate, thymidine -5'- phosphoric acid, uridine -5'- phosphorus
Acid, 6- phospho-fructoses, 6- phosphate-dextroses.
The catalase of the bacterium, beta galactosidase, arginine dihydrolase, gelatin enzyme reaction are the positive；Do not present urase,
Tryptophan deaminase, ornithine decarboxylase, lysine decarboxylase activity；Do not generate indoles and hydrogen sulfide；Nitrate reduction is in the moon
Property reaction.
Deposit number provided by the invention is the 16S rDNA gene sequences of the feed series bacillus of CGMCC NO.12912
The homology of the 16S rDNA sequences of row and each strain of bacillus genus is all very high, and maximum comparability reaches 99%, with
For Paenibacillus pabuli (AB073191.1) positioned at same cluster, affiliation is nearest.
The feed series bacillus that deposit number is CGMCC NO.12912 is coated on containing sea grass polysaccharide solid culture
On base tablet, cultivate 3~5 days.Obvious degradation circle can be formed by carrying out dyeing with Lugol's iodine solution.Therefore,
The present invention also provides the feed series bacillus that deposit number is CGMCC NO.12912 to prepare sea grass polysaccharide
Application in degrading enzyme.
The present invention also provides suitable for culture medium of the deposit number for the feed series bacillus of CGMCC NO.12912.
A kind of culture medium includes：
The carbon source is selected from alpha-cyclodextrin, beta-cyclodextrin, dextrin, starch, amarogentin, ursin, cellobiose, D- fruits
Sugar, D- galactolipins, gentiobiose, alpha-D-glucose, α-D- lactose, lactose, maltose, mannose, PEARLITOL 25C, D- sweet dews
Sugar, melezitose, melibiose, Alpha-Methyl-D- galactosides, Beta-methyl-D- galactosides, 3- methyl-glucose, Alpha-Methyl-D-
Glucoside, Beta-methyl-D-Glucose glycosides, Alpha-Methyl-D-MANNOSE glycosides, isomaltoketose, D-Psicose, D- raffinoses,
Salicin, D-glucitol, stachyose, sucrose, D-Tag, D- trehaloses, turanose, acetic acid, beta-hydroxybutyric acid, pyruvic acid first
Ester, pyruvic acid, monomethyl succinate, glycerine, adenosine, 2'- desoxyadenossines, inosine, thymidine, uridine, adenosine 5'-phosphate, chest
Glycosides -5'- phosphoric acid, uridine-5'-phosphate, 6- phospho-fructoses or 6- phosphate-dextroses；
Carbon source is selected from agarose, starch, sea grass polysaccharide, sucrose, maltose in the culture medium.In some specific embodiments,
The carbon source of the culture medium is sea grass polysaccharide.
A concentration of 0.4g/L~4g/L of carbon source.Specially 0.4g/L, 0.6g/L, 0.8g/L, 1g/L, 2g/L, 3g/L or
4g/L.Experiment shows that the concentration of carbon source influences the growing state of thalline and generates the vigor of enzyme, with the raising bacterium of carbon source concentration
The growing state of body is deteriorated, and enzyme activity then shows first to increase the trend reduced again with the raising of carbon source concentration.
Nitrogen source described in the culture medium is selected from NaNO3, urea, yeast extract, peptone.In the embodiment of the present invention, nitrogen
Source is peptone and yeast extract.In some specific embodiments, the yeast extract is yeast extract；The peptone is
Tryptone or casein peptone.The mass ratio of the peptone and yeast extract is (4~16)：(2~14).
In the embodiment of the present invention, a concentration of 4g/L~16g/L of peptone.Specially 4g/L, 6g/L, 8g/L, 10g/L,
12g/L, 14g/L or 16g/L.Experiment shows that the concentration of peptone influences the growing state of thalline and generates the vigor of enzyme, with
The growing state presentation of the raising thalline of peptone concentration first increases the trend reduced again, and peak appears in 10g/L, enzyme activity
Power shows first to increase the trend reduced again also with the raising of peptone concentration, is up to appear in 6g/L.
Similarly, a concentration of 3g/L~15g/L of yeast extract.Specially 3g/L, 5g/L, 7g/L, 9g/L, 11g/L,
13g/L or 15g/L.Experiment shows that the concentration of yeast extract influences the growing state of thalline and generates the vigor of enzyme, with yeast
The growing state presentation for soaking the raising thalline of powder concentration first increases the trend reduced again, and peak appears in 9g/L, enzyme activity
Raising also with yeast extract concentration shows first to increase the trend reduced again, is up to appear in 7g/L.
In the embodiment of the present invention, a concentration of 5g/L~35g/L of NaCl；Specially 5g/L, 10g/L, 15g/L, 20g/L,
25g/L, 30g/L or 35g/L.Experiment shows that the concentration of NaCl influences the growing state of thalline and generates the vigor of enzyme, with
The growing state of the raising thalline of NaCl concentration is deteriorated, and the vigor of enzyme also reduces.
In the embodiment of the present invention, Na2HPO4A concentration of 1mmol/L~9mmol/L；Specially 1mmol/L, 2mmol/L,
3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L or 9mmol/L.Experiment shows Na2HPO4It is dense
Degree influences the growing state of thalline and generates the vigor of enzyme, with Na2HPO4The growing state of the raising thalline of concentration is deteriorated, with
Na2HPO4The vigor of the raising enzyme of concentration first increases to be declined afterwards, and peak appears in 6mmol/L.
Experiments indicate that the pH value of culture medium influences thalli growth situation and generates the vigor of enzyme, the present invention is implemented
In example, the pH value of the culture medium is 6.0~8.0.Specially 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5 or 9.0.Thalline
Growth and enzyme activity peak appear in pH value for 7.0 when.
FePO4Hydrate be seven water ferric phosphates；MgSO4Hydrate be epsom salt.
The culture medium of verification the verifying results includes in embodiment：
Empirical tests, the preferable culture medium of effect include：
It is furthermore preferred that culture medium includes：
Culture medium provided by the invention is in culture deposit number is the feed series bacillus of CGMCC NO.12912
Using.
Agar is added in the culture medium can be made solid medium.
The mass fraction for adding agar is 1%~5%.
In order to activate strain provided by the invention, the present invention also provides a kind of culture mediums to include：
The pH value of the culture medium is 6.0~8.0, preferably 7.0.
Application of the culture medium in activation deposit number is the feed series bacillus of CGMCC NO.12912.
For activating in the culture medium of strain provided by the invention, the peptone is tryptone or casein peptone；Institute
Yeast extract is stated as yeast extract or yeast extract；Preferably yeast extract.
In some embodiments, the culture medium for activating strain provided by the invention includes
The present invention also provides a kind of method for preparing sea grass polysaccharide degrading enzyme, this method uses deposit number as CGMCC
Sea grass polysaccharide degrading enzyme is made in the feed series bacillus fermentation of NO.12912.
Specifically, the preparation of sea grass polysaccharide degrading enzyme includes：
Step 1：Using culture medium activation deposit number as the feed series bacillus of CGMCC NO.12912, seed is made
Liquid；
Step 2：Seed liquor is seeded to culture medium, fermented acquisition fermentate；
Step 3：Supernatant is taken after fermentate is centrifuged, after degerming, concentration, desalination, dry obtained sea grass polysaccharide degradation
Enzyme.
The step of pre-treatment being further included before activation described in step 1；It by deposit number is CGMCC that the pre-treatment, which is,
The feed series bacillus of NO.12912 is in scribing line culture 5 days~7 days on the solid medium containing sea grass polysaccharide.
The feed series bacillus that deposit number is CGMCC NO.12912 is activated in the embodiment of the present invention, in step 1
Culture medium includes：
Culture medium is provided by the present invention used by fermenting in step 2, including：
The culture medium that pre-treatment uses is to add agar in the culture medium used in fermentation, forms it into solid medium,
Specifically, solid medium includes：
The condition activated described in step 1 is 24 DEG C~36 DEG C, shaking table culture 12~for 24 hours；The rotating speed of the shaking table is 150
~210rpm.
The condition fermented described in step 2 is 24 DEG C~36 DEG C, 24~48h of shaking table culture；The rotating speed of the shaking table is 150
~210rpm.
Fermentation temperature, rotating speed, inoculum concentration and fermentation volume are screened in embodiment.
Wherein, fermentation temperature is 20 DEG C, 24 DEG C, 28 DEG C, 32 DEG C, 36 DEG C.The result shows that 28 DEG C are most suitable fermentation temperature.
The rotating speed of fermentation is 40rpm, 80rpm, 120rpm, 160rpm, 200rpm, 240rpm.The result shows that 200rpm is
Most suitable rotating speed.
Inoculum concentration is 1%~8%, specially 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%.Take off the result shows that, most
Suitable inoculum concentration is 3%.
Fermentation volume be 50mL~120mL, specially 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 110mL,
120mL.The result shows that most suitable fermentation volume is 70mL.
The present invention research shows that, under suitable condition, for 24 hours fermentation gained sea grass polysaccharide degrading enzyme activity and amount i.e. can reach
Higher level.
The rotating speed centrifuged described in step 3 is 6000rpm, time 20min.
Centrifugation, degerming, concentration, desalination described in step 3 are all in 4 DEG C of progress.
The degerming uses filtration sterilization, and filter diameter is 0.22 μm.
Concentration is using ultrafiltration concentration.
Desalination is using dialysis desalination.
The aperture of dialysis is 8000~14000Da
The component of dialyzate includes：0.5mMPBS buffer solutions
Dry is vacuum freeze drying；Program is -80 DEG C of freezings, and 48h is lyophilized in -50 DEG C of freeze-dryings.
The present invention also provides using deposit number as waterside made from the feed series bacillus fermentation of CGMCC NO.12912
Tongue polysaccharide degrading enzyme.
The present invention provides deposit number be CGMCC NO.12912 feed series bacillus, the present invention provides for
Screen culture medium, the culture medium for activated spawn and the culture medium for fermentation of strain.And it provides with the strain fermentation
Prepare sea grass polysaccharide degrading enzyme method and fermentation made from sea grass polysaccharide degrading enzyme.It is produced with strain provided by the invention and fermentation
Raw sea grass polysaccharide degrading enzyme has good enzymatic activity, and can degrade sea grass polysaccharide, and can be prepared not according to different parameters
With the sea grass polysaccharide oligosaccharides of the degree of polymerization.Experiment shows the activity of sea grass polysaccharide degrading enzyme provided by the invention up to 1.03U/mL.
Its sea grass polysaccharide oligosaccharides (low molecular weight sea grass polysaccharide) molecular weight obtained is 4.48 × 104Da；Monosaccharide forms：Rhamnose
(Rha)：Glucose (Glc)：Galactolipin (Gal)：Xylose (Xyl)：Arabinose (Ara)=1.65：1.00：0.09：0.57：
0.17。
Biological deposits explanation
EP-1, Classification And Nomenclature：Feed series bacillus Paenibacillus pabuli, in preservation on the 29th in 08 month in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address is：Chaoyang District, Beijing City North Star west
No. 3 Institute of Microorganism, Academia Sinica of institute of road 1.Deposit number is CGMCC No.12912.
Description of the drawings
The scanning electron microscope (SEM) photograph of Fig. 1 bacterial strains EP-1 (deposit number is CGMCC No.12912)；
The phylogenetic tree of Fig. 2 bacterial strains EP-1 (deposit number is CGMCC No.12912) and its close bacterial strain；
Fig. 3 shows sea grass polysaccharide superoxide radical scavenging capacity；
Fig. 4 shows sea grass polysaccharide hydroxyl radical free radical scavenging capacity；
Fig. 5 shows sea grass polysaccharide DPPH free radical scavenging activities；
Fig. 6 shows sea grass polysaccharide reducing power；
Fig. 7 shows the metal chelation abilities of sea grass polysaccharide.
Specific embodiment
The present invention provides a kind of feed series bacillus, its culture medium and answering in sea grass polysaccharide degrading enzyme is prepared
With those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.It is in particular, it should be pointed out that all
Similar replacement and change is apparent to those skilled in the art, they are considered as being included in the present invention.This
The method of invention and application are described by preferred embodiment, and related personnel can be not significantly being departed from the present invention
Hold, methods herein and application be modified or suitably changed with combining in spirit and scope, to realize and using the present invention
Technology.
The instrument that the present invention uses is all common commercially available product, can all be bought in market.
With reference to embodiment, the present invention is further explained：
Embodiment 1
Sea grass polysaccharide degradation bacteria (Paenibacillus pabuli) EP-1 is isolated and purified.
The ooze of Qingdao of Shandong province No.One Bathing Beach surrounding waters is acquired, antiseptic sea water dissolving is added in, moves into primary dcreening operation
In culture medium, shaking flask enrichment culture 2-3 days.Culture solution is coated on containing on sea grass polysaccharide solid medium tablet, cultivates 3-5
My god.It is dyed with Lugol's iodine solution, by the colony lift for having obvious degradation circle to new sea grass polysaccharide solid medium tablet
Upper culture repeats to cross, until obtaining pure culture.Primary dcreening operation medium component (mass volume ratio)：Sea grass polysaccharide 4%,
NaCl1.5%, NaNO30.5%, MgSO4·7H2O0.05%, CaCl20.01%, ferric phosphate 0.005%, pH value 7.0.
The pure culture that primary dcreening operation is separated to is inoculated into fermentation medium, shaking table culture.Then by culture solution in Enteromorpha
It crosses on polysaccharide solid medium, the bacterium colony picking for generating most degradation circle is out subjected to next step experiment.It is all to filter out
The bacterial strain come conservation after isolating and purifying.
Form and the physiology characteristic identification of bacterial strain (Paenibacillus pabuli.sp) EP-1 is used for the bacterial strain of identification
EP-1 is inoculated in sea grass polysaccharide culture medium, and the agar of 15g/l, 121 DEG C of sterilizings can be added when preparing solid medium
30min。
Single bacterium colony is obtained through plate streaking culture, which has following morphological feature：
(1) colonial morphology：Good in sea grass polysaccharide cultured on solid medium, bacterium colony milky is round, and surface is slightly convex
It rises, smooth, non-reflective, opaque, neat in edge, diameter is about 3.0mm；
(2) cellular morphology：Cell is in direct rod shape, 1.8-3.5 μm of 0.6-0.8 μ ms, single arrangement, Gram's staining sun
Property, uniform coloring can produce pod membrane, move (peritrichous)；It is raw or near middle raw in gemma, it is wide less than or equal to cell, it is rounded
Or ellipse.
The amplification of the 16S rRNA genes of bacterial strain (Paenibacillus pabuli.sp) EP-1, sequence and analysis
Bacterial strain EP-1 lines sea grass polysaccharide culture medium slant, 28 DEG C of cultures to formation lawn.Aseptically use oese
It scrapes a ring thalline to be placed in 1.5mL sterile centrifugation tubes, genomic DNA is extracted using bacterial genomes Rapid extraction kit,
PCR is directly used in as template.The universal primer for expanding 16S rRNA genes is as follows：
Forward primer：5'-AGAGTTTGATCCTGGCTCAG-3'；
Reverse primer：5'-ACGGTTACCTTGTTACGACTT-3'；
Above-mentioned primer corresponds to 8~27 and 1510~1492 bit bases of Escherichia coli 16S rRNA genes respectively.50μL
PCR reaction systems are：10 × buffer, 5 μ L, 10mM dNTPs 1 μ L, 4 μM of primers each 1 μ L, ddH242 μ L, Taq DNA of O gather
0.5 μ L (2.5U) of synthase, 0.5 μ L (10-100ng) of DNA profiling.PCR reaction conditions are：94 DEG C of pre-degeneration 5min；95 DEG C of denaturation
30s, 55 DEG C of annealing 30s, 72 DEG C of extension 75s；Extend 10min after 33 cycles after 72 DEG C, the PCR product sequencing of acquisition by
Sangon Biotech (Shanghai) Co., Ltd. completes.The sequence length of the 16S rRNA genes of obtained bacterial strain EP-1 is
1372bp。
Obtained sequence carries out similarity system design using Blast programs.With MEGA (Molecular Evolutionary
Genetics Analysis) 6 software packages, with adjacent method (neighbor-joining method) to each of bacillus genus
The structure (Fig. 2) and tetraploid rice of a kind and EP-1 progress systematic evolution tree.It was found that EP-1 bacterial strain 16SrRNA gene orders with
The homology of the 16S rRNA sequences of each kind of bacillus genus is all very high, and maximum comparability reaches 99%.Chadogram shows
Show EP-1 and Paenibacillus pabuli (AB073191.1) positioned at same cluster, affiliation is nearest, will be at the beginning of EP-1 bacterial strains
Step is accredited as feed series bacillus.
Bacterial strain EP-1 is preserved in CGMCC, deposit number is CGMCC NO.12912.
Embodiment 2
Be respectively configured culture medium shown in table 1, deposit number for CGMCC NO.12912 feed series bacillus it is activated after,
It is seeded to 1 culture medium of table.Inoculum concentration is 5%, and cultivation temperature is 28 DEG C, shaking speed 200rpm, and culture for 24 hours, measures bacterium solution
OD600Value.
Gained bacterium solution 6000rpm centrifugations 20min obtains fermented supernatant fluid；Under the conditions of 4 DEG C, gained fermented supernatant fluid is crossed 0.22
μm filter membrane degerming, ultrafiltration concentration, dialysis desalination, vacuum freeze drying, obtain sea grass polysaccharide degrading enzyme.Measure enzyme activity.Enzyme activity
Detection method for DNS methods, enzyme activity definition：1mL enzyme solutions 1min generates 1 μ g reduced sugars as a unit of activity.
Table 1, culture medium prescription
According to table 1, optimum carbon source is sea grass polysaccharide, and optimum nitrogen source is peptone and the composition of dusty yeast.Suitable condition
Under, the enzyme activity of sea grass polysaccharide degrading enzyme is up to 1.03U/mL.
Embodiment 3
The culture medium of best results in table 1 is used (to be formulated as sea grass polysaccharide 0.2%, peptone 0.6%, yeast extract
0.7%, NaCl 0.5%, MgSO4·7H2O 0.1%, Na2HP04It 6mmol/L) carries out fermentation and prepares sea grass polysaccharide degrading enzyme.
Through pre-treatment and activation before fermentation.
1), pre-treatment：The feed series bacillus that deposit number is CGMCC NO.12912 is lined sea grass polysaccharide to consolidate
On body culture medium, 5~7d is cultivated at 25~35 DEG C；
2) it, activates：Solid medium bacterial strain is connect into 1~3 ring in the test tube of seed culture fluid, at 25~35 DEG C with
150~210rpm cultures 12~for 24 hours.
3) it, ferments：Seed culture medium is accessed in fermentation medium, with 24~48h of shaking table culture, obtains fermentate.
Step 3 carries out the fermentation test of different parameters, each parameter such as table 2：
After fermentation, bacterium solution OD is measured600Value.
Gained bacterium solution 6000rpm centrifugations 20min obtains fermented supernatant fluid；Under the conditions of 4 DEG C, gained fermented supernatant fluid is crossed 0.22
μm filter membrane degerming, ultrafiltration concentration, dialysis desalination, vacuum freeze drying, obtain sea grass polysaccharide degrading enzyme.Measure enzyme activity.Enzyme activity
Detection method for DNS methods, enzyme activity definition：1mL enzyme solutions 1min generates 1 μ g reduced sugars as a unit of activity.
2 fermentation condition of table
The result shows that inoculum concentration 3%, cultivation temperature is 28 DEG C, shaking speed 200rpm, is cultivated for 24 hours, fermentation volume
70mL, generated enzyme activity highest.
Embodiment 4
Preparing low molecular weight sea grass polysaccharide with sea grass polysaccharide degrading enzyme made from embodiment 2 or embodiment 3, (sea grass polysaccharide is few
Sugar.)
Enteromorpha dry powder is cleaned with tap water and (removed desalination and other impurities), is dried for standby in 60 DEG C.Use Hot water extraction
Polysaccharide is carried, by Enteromorpha：Water (w/v)=1:30 add in distilled water, and 100 DEG C of heating stirrings extract 2h, with 200 mesh silk cover filterings, filter
Slag repeats to extract once, merges coarse filtration liquid twice, measures volume.Coarse filtration liquid placement is cooled to gel, cuts after -20 DEG C of refrigerators
Freezing.It takes out, break into pieces after the complete deep colling of adhesive tape, add in 95% alcohol of 1.5 times of coarse filtration liquid volumes, be placed at room temperature for, make ice cube
It thaws dehydration.With 200 mesh silk cover filterings, water and alcohol are squeezed with hand.Filter residue is further taken off with a certain amount of 95% alcohol again
Water then in 60 DEG C of drying, obtains Enteromorpha Thick many candies.
Thick many candies are isolated and purified using DEAE Sepharose Fast Flow Weak anion-exchange chromatography columns, are used
Fraction collector collects fraction, and total sugar content is detected using sulfuric acid-phynol method.According to linear elution as a result, using distilled water respectively
2 column volumes are eluted, 0.5mol/L NaCl elute 2 column volumes, and 1.07mol/L NaCl elute 3 column volumes, and 4mol/L is washed
It takes off 2 column volumes, then 6 column volumes is rinsed with deionized water (purpose is to remove extra salting liquid)；Column volume：60mL,；Loading
Amount：30mg；Flow velocity：3.5mL/min.Fraction collector is collected, sulfuric acid-phynol method detection total sugar content, with elution volume, extinction
Degree and salinity mapping.Distilled water elution fraction is collected, dialysis desalination, concentration, freeze-drying are placed in drier and preserve.
Through the isolated polysaccharide component of DEAE Sepharose Fast Flow anion-exchange chromatographies, continue through
Sepharose 6B gel chromatographic columns (2.6 × 80cm) are further isolated and purified.Using water as eluent, flow velocity 1mL/
min.Component (10mL/ pipes) is collected using fraction collector；Total sugar content is detected with sulfuric acid-phynol method, is surveyed at 490nm wavelength
Its fixed absorption value, is mapped with absorbance-elution time.Merge same composition through dialysis desalting, freeze-drying, be placed in drier and protect
It deposits.
The preparation of low molecular weight sea grass polysaccharide passes through enzymatic hydrolysis process.By sea grass polysaccharide degrading enzyme and sea grass polysaccharide (enzyme
Measure 2% for polysaccharide quality) 72h is reacted in 45 DEG C, boiling water boils 10min, makes protein inactivation, and 8000rpm centrifugation 10min are received
Collect the unreacted polysaccharide of ethanol precipitation removal that supernatant adds in 95%.Supernatant is concentrated through rotary evaporation, and dialysis (≤
2000Da), it is freeze-dried, obtains low molecular weight sea grass polysaccharide.
After testing, low molecular weight sea grass polysaccharide color is white, soluble easily in water, and total sugar content 56.08 ± 1.19% has no
Uronic acid, sulfate content are 17.934 ± 0.12%, protein content 0.64 ± 0.14；Molecular weight is 4.48 × 104Da；Monosaccharide
It forms and is：Rhamnose (Rha)：Glucose (Glc)：Galactolipin (Gal)：Xylose (Xyl)：Arabinose (Ara)=1.65：
1.00：0.09：0.57：0.17.
Further measure the bioactivity of gained low-molecular-weight polysaccharide, the results showed that, superoxide radical scavenging capacity (figure
3), hydroxyl radical free radical Scavenging activity (Fig. 4), DPPH free radical scavenging abilities (Fig. 5), reproducibility (Fig. 6), metal chelation abilities
(Fig. 7) is all relatively strong.
It the above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (12)
- A kind of 1. screening and culturing medium, which is characterized in that the component including water and following mass fraction：
- 2. application of the screening and culturing medium described in claim 1 in screening can generate the bacterial strain of sea grass polysaccharide degrading enzyme.
- 3. deposit number is the feed series bacillus of CGMCC NO.12912.
- 4. deposit number is application of the feed series bacillus of CGMCC NO.12912 in sea grass polysaccharide degrading enzyme is prepared.
- 5. a kind of fermentation medium, which is characterized in that including：The carbon source is selected from agarose, starch, sea grass polysaccharide, sucrose, maltose；The nitrogen source is selected from NaNO3, urea, yeast extract, peptone.
- 6. the answering in culture deposit number is the feed series bacillus of CGMCC NO.12912 of culture medium described in claim 5 With.
- 7. a kind of seed culture medium, which is characterized in that including：
- 8. the culture medium described in claim 7 is in activation deposit number is the feed series bacillus of CGMCC NO.12912 Using.
- A kind of 9. method for preparing sea grass polysaccharide degrading enzyme, which is characterized in that by the feeding that deposit number is CGMCC NO.12912 Expect that sea grass polysaccharide degrading enzyme is made in series bacillus fermentation.
- 10. according to the method described in claim 9, it is characterized in that, the preparation of the sea grass polysaccharide degrading enzyme includes：Step 1：Using the culture medium activation deposit number described in claim 7 as the feed class gemma bar of CGMCC NO.12912 Seed liquor is made in bacterium；Step 2：The seed liquor is seeded to the culture medium described in claim 5, fermented acquisition fermentate；Step 3：Supernatant is taken after the fermentate is centrifuged, after degerming, concentration, desalination, dry obtained sea grass polysaccharide degradation Enzyme.
- 11. according to the method described in claim 9, it is characterized in that, the step of further including pre-treatment before activation described in step 1； The pre-treatment is to train the feed series bacillus that deposit number is CGMCC NO.12912 in the solid containing sea grass polysaccharide Support scribing line culture 5 days~7 days on base.
- 12. sea grass polysaccharide degrading enzyme made from any one of claim 9~11 the method.
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