CN101440355A - Enterobacter cloacae, and use of polysaccharide and polysaccharide thereof - Google Patents
Enterobacter cloacae, and use of polysaccharide and polysaccharide thereof Download PDFInfo
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Abstract
The invention discloses Enterobacter cloacae Z0206, which has been preserved in China General Microbiological Culture Collection Center on December 3, 2007, wherein the preserving number is CGMCC NO: 2279. The invention also discloses a method for preparing Enterobacter cloacae Z0206 polysaccharide and application thereof. The method has the advantages that the production process of extracting the polysaccharide is simple, and the yield and the purity of products are higher; the Enterobacter cloacae Z0206 polysaccharide can remarkably improve the proliferation capacity of lymph cells and the cytokine level of a small mouse with low immunity and has remarkable adjustment effect on the immunologic function of the small mouse with low immunity; and the Enterobacter cloacae Z0206 polysaccharide has apparent antagonism on the oxidative damage caused by cyclophosphamide, can remarkably improve the antioxidase activity of an organism, strengthen the function of an antioxidant defense system of the organism, and prevent harmful free radicals from damaging biological macromolecules in cells.
Description
Technical field
The present invention relates to bioengineering field, design a kind of preparation method and application thereof especially with anti-oxidant, antitumor, strengthening immunity and bioactive enterobacter cloacae Z0206 bacterial polysaccharides such as antiviral.
Background technology
Polysaccharide is very wide in distributed in nature, and existence is all arranged in higher plant, animal, microbe, is one of the abundantest biological polymer of nature content.Polysaccharide has many-sided function, as energy storage, structural support, defense function etc.As far back as the forties in last century, polysaccharide just begins as medicine, nineteen fifty-one American Reihy H finds that first the polysaccharide in the microorganism basidiomycetes has the activity that suppresses tumour, to the sixties, polysaccharide conduct immunopotentiating agent widely causes people's very big interest, and the exploitation polysaccharide is that protective foods and medicine also receive much attention.Have and studies have shown that adding active polysaccharide in the feed can significantly improve the animal immune function, improve growth of animal and production performance, reduce the animal dead rate.
A lot of polysaccharide have immune-enhancing activity, show mediation and regulating effect to host immune, by maturation, differentiation and the propagation of immune stimulatory cell, improve host's organism balance, reach recovery and improve the reactivity of host cell lymphokine, hormone and other physiological factors.Based on this, polysaccharide shows the various active function relevant with immunizing power, as synthetic, the anti-inflammatory of antitumor, hypoglycemic, anti-ageing, as to promote liver and medullary cell protein and nucleic acid and radiation resistance etc.
In recent years, we separate the bacterial isolates enterobacter cloacae Z0206 that has obtained plant height product exocellular polysaccharide by separation, screening and purifying from Ganoderma.So-called Ganoderma among the people being referred to as " local tyrant ", is put down in writing according to Compendium of Material Medica, Ganoderma is that book on Chinese herbal medicine is top grade, it is a kind of natural rare species of finding in China, and promptly a kind of protoplasma life entity of being assert already by China scientific circles is definitely named and be should be " super-huge Acarasiales complex body ".Studies show that bacterial strain enterobacter cloacae Z0206 has the characteristic of high yield polysaccharide, this bacterial polysaccharides arrives outside the born of the same parents with the excretory form, have the output height, safely, advantage such as have no side effect.Enterobacter cloacae Z0206 bacterial polysaccharides has certain anti-oxidant, antitumor, antiviral and immunomodulatory isoreactivity.Therefore, the preparation of enterobacter cloacae Z0206 bacterial polysaccharides has great importance and wide prospect in anti-oxidant and antitumor drug and additive development and application.
Summary of the invention
A kind of enterobacter cloacae Z0206 of the present invention, being preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 3rd, 2007 (is called for short: CGMCC) (Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preserving number: CGMCC NO:2279.Classification name: enterobacter cloacae Enterobacter cloacae.
A kind of preparation method of enterobacter cloacae Z0206 polysaccharide may further comprise the steps successively:
(1) enterobacter cloacae Z0206 actication of culture: the enterobacter cloacae Z0206 bacterial classification of the present invention that takes out freezing preservation, after room temperature is placed 1h, being inoculated into sterilization PDA adds on the rich solid medium, cultivate 36h～48h for 28 ℃～32 ℃, being transferred to PDA once more adds on the rich solid medium, cultivate 36h～48h, obtain activated spawn for 28 ℃～32 ℃;
(2) will activate good bacterial classification, being seeded to PDA adds and carries out shake flask fermentation in the liquid-rich substratum, 28 ℃～32 ℃ reciprocating vibrations are cultivated, incubation time 18h～20h obtains the ferment tank seed liquor, adds 70% fermention medium in the fermentor tank, 121 ℃ of steam sterilizing 20min, the inoculation seed liquor, fermentation time 48h～72h obtains containing the fermented liquid of enterobacter cloacae Z0206 polysaccharide;
(3) fermented liquid is concentrated into 1/10 of original volume in 60 ℃～70 ℃, 80 ℃ again～90 ℃ water-bath 1h～1.5h, centrifugal 10min～the 15min of 5000rpm collects supernatant liquor, adds 95% ethanol of 3～5 times of volume precoolings, 4 ℃ leave standstill 12h after, centrifugal 10min～the 15min of 5000rpm, precipitation is more successively with dehydrated alcohol, acetone and anhydrous diethyl ether washing, and is centrifugal, precipitation vacuum-drying promptly obtains the Crude polysaccharides powder;
(4) protease treatment: the Crude polysaccharides that step (3) is obtained is dissolved in the distilled water of heat of 20 times of volumes, uses Na
2CO
3Transfer pH to 7.0～9.0, add 0.25%～1% trypsin w/v), behind 55 ℃ of hydrolysis 1～2h, transfer pH to 5.0～5.7 with oxalic acid, add 0.25%～1% papoid (w/v) then, behind 65 ℃～70 ℃ hydrolysis 2h～3h, in 100 ℃ of heating in water bath 5～6min, to stop enzyme reaction;
(5) trichloroacetic acid method deproteinated: get the protease treatment liquid that obtains after the step (4), with oxalic acid adjust pH to 7.0, add 2%～4% trichoroacetic acid(TCA) (w/v), after the room temperature lower magnetic force stirred 1h, the centrifugal 10～15min of 11000rpm got supernatant;
(6) Sevag method deproteinated: add the Sevag reagent of 1/3 volume in the supernatant liquor that step (5) obtains, fully vibration mixes 20～30min, the sufficient standing layering, and the centrifugal 5min of 4000rpm, albumen precipitation no longer appears in repetitive operation several to two-phase interface; Use 95% alcohol chromatography of 3～5 times of volume precoolings then, the precipitation of gained is used dehydrated alcohol, acetone and anhydrous diethyl ether washed twice more successively, and vacuum-drying obtains the deproteinated polysaccharide;
(7) decolouring: will be made into 0.5% polysaccharide soln through the polysaccharide behind step (6) deproteinated, and transfer pH to 8.0, and under 50 ℃, drip 20% H with ammoniacal liquor
2O
2, be faint yellow to solution, insulation 2h is neutralized to 7.0 with dilute hydrochloric acid then; Tap water dialysis 2～3d behind redistilled water dialysis 2～3d, adds 95% ethanol sedimentation of 3～5 times of volume precoolings, 4 ℃ leave standstill 12h after, the centrifugal 10min～15min of 5000rpm, precipitation vacuum-drying, polysaccharide products.
In described step (1) and (2), the prescription of PDA enriched medium is: potato 20%, peptone 0.2～0.5%, yeast extract 0.3%, glucose 2%, agar powder 1.6%; The prescription of fermention medium is: sucrose 2.5%, yeast extract 0.5%, peptone 0.5%, K
2HPO
40.2%, KH
2PO
40.1%, MgSO
40.05%.
In the described step (2), conditions of flask fermentation is: inoculum size 3～5 rings, reciprocating vibration stroke 4～10cm, oscillation frequency 200～250rpm.
In the described step (2), the ferment tank condition is: inoculum size 3%, and pH is 7.0,30 ℃ of temperature, air flow is 0.25～0.75vvm, mechanical stirring rotating speed 200～300rpm, every 12h replenish 1%～3% glucose to fermentor tank.
The prescription of the Sevag reagent in the described step (6) is that the volume ratio of chloroform and propyl carbinol is 4: 1.
A kind of enterobacter cloacae Z0206 polysaccharide is in preparation enhancing body anti-oxidant function and the healthcare products of immunoloregulation function or the application in the additive.
The application of a kind of enterobacter cloacae Z0206 polysaccharide in preparation antitumor drug and antiviral.
Advantage of the present invention:
(1) adopt phenolsulfuric acid method and Kjeldahl determination to measure polysaccharide and protein content in the enterobacter cloacae Z0206 polysaccharide.Measurement result shows: polysaccharide, protein content are respectively in the Crude polysaccharides: 68.02%, 28.41%; Polysaccharide, protein content are respectively in the polysaccharide products: 99.6% and 0.03%.The result shows that the production process of said extracted polysaccharide is simple, and product production and purity are higher.
(2) enterobacter cloacae Z0206 polysaccharide can significantly improve immunocompromised mouse lymphocyte multiplication capacity and cytokine levels, and the immunocompromised immune function of mice is had significant regulating effect.
(3) enterobacter cloacae Z0206 polysaccharide has tangible antagonistic action to the oxidative damage by caused by cyclophosphamide, can significantly improve the body activities of antioxidant enzymes, the function of enhancing body anti-oxidative defense system prevents the damage of biomacromolecule in harmful radical pair cell.
Embodiment
Below in conjunction with specific examples technical scheme of the present invention is described further:
The preparation method of embodiment 1, enterobacter cloacae Z0206 polysaccharide
(1) actication of culture: PDA enriched medium prescription is as follows: potato 20%, peptone 0.3%, yeast extract 0.3%, glucose 2%, agar powder 1.6%.Cultivate flat board and 121 ℃ of sterilizations of PDA enriched medium 20min of usefulness, fall dull and stereotyped at sterilisable chamber, treat after the substratum cooled and solidified, get the enterobacter cloacae Z0206 bacterial classification of freezing preservation, be inoculated on the PDA enriched medium flat board with transfering loop, cultivate 2d for 30 ℃, repetitive operation once obtains activatory enterobacter cloacae Z0206 bacterial classification.
(2) preparation of enterobacter cloacae Z0206 polysaccharide fermentation liquid: in the 250ml triangular flask, add the above-mentioned PDA of 70ml and add the liquid-rich substratum, 121 ℃ of sterilization 20min, after inoculation of medium 5 ring activated spawn, be positioned over 30 ℃ of shaking culture in the shaking table, the stroke 4-10cm of reciprocating vibration, oscillation frequency 200rpm obtains the fermentor tank seed liquor behind the cultivation 18h.(prescription of fermention medium is: sucrose 2.5%, yeast extract 0.5%, peptone 0.5%, K to add 70% fermention medium in the fermentor tank
2HPO
40.2%, KH
2PO
40.1%, MgSO
40.05%), 121 ℃ of steam sterilizing 20min, the inoculation seed liquor, inoculum size 3%, fermentation condition is that pH is 7.0,30 ℃ of temperature, air flow are 0.5vvm, mechanical stirring rotating speed 200rpm, every 12h replenishes 1% glucose to fermentor tank, and fermentation time 48h obtains containing the fermented liquid of enterobacter cloacae Z0206 polysaccharide.
(3) separation and Extraction of enterobacter cloacae Z0206 polysaccharide:
A. the extraction of Crude polysaccharides
The fermented liquid Rotary Evaporators, in 60 ℃ of 1/10,90 ℃ of water-bath 1h, the centrifugal 15min of 5000rpm with its simmer down to original volume, collect supernatant liquor, in supernatant liquor, slowly add the cold dehydrated alcohol of 3 times of volumes while stirring with magnetic stirring apparatus, put into 4 ℃ of refrigerators precipitations and spend the night, then the centrifugal 15min of 5000rpm, precipitation is washed with the acetone and the anhydrous diethyl ether of precooling successively, centrifugal, at last precipitation is placed vacuum drier dry, obtain the Crude polysaccharides powder.
B. protease treatment:
Get Crude polysaccharides 10g and be dissolved in (heat is short molten a little) in the 200mL distilled water, use Na
2CO
3Transfer pH value to 8.0, add trypsinase 0.5g, behind 55 ℃ of hydrolysis 2h, transfer pH value to 5.5 with oxalic acid, add papoid 0.5g, behind 70 ℃ of hydrolysis 3h, be heated to 100 ℃, 5min stops enzyme reaction.
C. trichloroacetic acid method deproteinated:
Get the protease treatment liquid of 200mL, transfer pH value to 7.0 with oxalic acid, add the 8g trichoroacetic acid(TCA), use magnetic stirrer 1h, the centrifugal 10min of 11000rpm gets supernatant.
D.Sevag method deproteinated:
Get in the above-mentioned supernatant liquor, add the Sevag reagent (volume ratio of chloroform and propyl carbinol is 4:1) of 1/3 volume, abundant vibration mixes 25min, the sufficient standing layering, and the centrifugal 5min of 4000rpm repeats for several times till two-phase interface does not have the metaprotein appearance.Use 95% alcohol chromatography of 3 times of volume precoolings then, the precipitation of gained is used dehydrated alcohol, acetone and anhydrous diethyl ether washed twice more successively, and vacuum-drying obtains the deproteinated polysaccharide.
E. decolouring:
Polysaccharide behind the deproteinated is made into 0.5% polysaccharide soln, transfers pH value to 8.0, drip 20% H down at 50 ℃ with ammoniacal liquor
2O
2, be faint yellow to solution, insulation 2h is neutralized to 7.0 with dilute hydrochloric acid then.Tap water dialysis 48h behind the distill water dialysis 48h, adds 3 times of volume precoolings and gets 95% ethanol, 4 ℃ leave standstill 12h after, the centrifugal 10min of 5000rpm, precipitation vacuum-drying must polysaccharide products.
Embodiment 2, application phenolsulfuric acid method detect polysaccharide content in the enterobacter cloacae Z0206 polysaccharide
Method is as follows: accurately take by weighing standard glucose 20mg in the 500ml volumetric flask, add water to scale, absorption 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml, 1.8ml add water respectively and mend to 2.0ml, add 6.0% phenol 1.0ml and vitriol oil 5.0ml then, leave standstill 10min, shake up, the 30min water-bath, measure optical density(OD) in 490nm behind the 20min, with 2.0ml water by being operating as blank equally, X-coordinate x is a polysaccharide content, and ordinate zou y is an optical density value, the drawing standard curve.Draw sample liquid 1.0ml (being equivalent to the polysaccharide about 40 μ g),, measure optical density(OD), calculate polysaccharide content with typical curve by the above-mentioned steps operation.
Embodiment 3, application Kjeldahl determination are measured protein content in the enterobacter cloacae Z0206 polysaccharide.
Adopt the Kjeldahl determination (GB/T 5009.5-1985) of national Specification to measure protein content in the enterobacter cloacae Z0206 polysaccharide.
Embodiment 2 and embodiment 3: adopt phenolsulfuric acid method and Kjeldahl determination to measure polysaccharide and protein content in the enterobacter cloacae Z0206 polysaccharide.Measurement result shows: polysaccharide, protein content are respectively in the Crude polysaccharides: 68.02%, 28.41%; Polysaccharide, protein content are respectively in the polysaccharide products: 99.6% and 0.03%.The result shows that the production process of said extracted polysaccharide is simple, and product production and purity are higher.
Embodiment 4, enterobacter cloacae Z0206 polysaccharide immunoregulatory activity and anti-oxidant activity research.
Get 40 of ICR mouse (male and female half and half), be divided into control group (I), endoxan group (II), endoxan+polysaccharide group (III) and polysaccharide group (IV) at random, I, II organize and irritate stomach physiological saline 0.4mL every day, III, IV organize and irritate stomach polysaccharide 0.4mL (400mg/kg body weight) every day, II, III group is low at 12d intraperitoneal injection of cyclophosphamide (50mg/kg body weight) induction of immunity, trial period 14d, at the 10th day of test, the SRBC of all test mouse abdominal injection 0.2mL 10% carried out immunity.After last administration 12 hours, mouse was put to death in the cervical vertebra dislocation, and following index analysis is carried out in sampling.
1, gather spleen, mtt assay carries out the spleen lymphocyte proliferation test, measures T/B lymphopoiesis effect:
After the off-test, put to death mouse, with 75% alcohol-pickled 5min, get spleen (aseptic technique), place Hank ' S liquid to grind in 200 order nets, sterile preparation becomes single cell suspension, and adjusting cell concn with the RPMI-1640 nutrient solution is 2 * 10
6/ ml, the every hole of 96 porocyte culture plates adds the mouse boosting cell suspension 90 μ L of different treatment respectively, the ConA liquid 10 μ L of 50 μ g/mL or the LPS liquid 10 μ L of 100 μ g/mL, control wells adds splenocyte suspension and the 10 μ L RPMI-1640 nutrient solutions of 90 μ L, put 5%CO2,37 ℃ of cell culture incubators leave standstill cultivation, in cultivating 68h, take out culture plate, every hole adds MTT (0.4%) 10 μ L, every hole adds 100 μ L DMSO after continuing to cultivate 4h, shakes up with dull and stereotyped shaking table, and purple crystal dissolves the back fully surveys the OD value with enzyme-linked immunosorbent assay instrument (BIO-RAD) in the 570nm place.
2, blood sample collection prepares serum, adopts ELISA method kit measurement Cytokine of Serum IL-2 and TNF-alpha content.Gather liver samples, prepare 10% liver homogenate, adopt SOD and GSH-Px activity in the kit measurement liver.
Test-results is as shown in the table:
Table 1 is the influence of enterobacter cloacae Z0206 polysaccharide to ICR mouse spleen lymphocyte propagation;
Table 2 is the influence of enterobacter cloacae Z0206 polysaccharide to ICR mice serum cytokine IL-2 and TNF-alpha content;
Table 3 is that enterobacter cloacae Z0206 polysaccharide is to the active influence of SOD and GSH-Px in the ICR mouse liver.
Table 1
Annotate: same column subscript letter different table differential different significantly (p＜0.05).
Compare with control group, behind the mouse peritoneal injection endoxan, ConA inductive splenic T lymphocytic proliferation rate and LPS inductive bone-marrow-derived lymphocyte proliferation rate reduce by 32.16% (p＜0.05) and 43.37% (p＜0.05) respectively; Compare with endoxan effect group separately, mouse is through the preventative filling stomach of 400mg/kg polysaccharide intraperitoneal injection of cyclophosphamide again, and ConA inductive splenic T lymphocytic proliferation rate and LPS inductive bone-marrow-derived lymphocyte proliferation rate improve 10.14% (p＜0.05) and 22.68% (p＜0.05) respectively.
Table 2
Annotate: same column subscript letter different table differential different significantly (p＜0.05).
Compare with control group, behind the mouse peritoneal injection endoxan, IL-2 and TNF-alpha levels reduce by 40.04% (p＜0.05) and 47.41% (p＜0.05) respectively in the serum; Compare with endoxan effect group, mouse is through the preventative filling stomach of 400mg/kg polysaccharide intraperitoneal injection of cyclophosphamide again, and the TNF-alpha levels improves 34.30% (p＜0.05) in the serum; Compared with the control, irritate separately that the TNF-alpha levels improves 7.84% in the polysaccharide group serum of stomach 400mg/kg, but difference not significantly (p〉0.05).
Table 3
Annotate: same column subscript letter different table differential different significantly (p＜0.05).
Compare with control group, behind the mouse peritoneal injection endoxan, SOD and GSH-Px activity reduce by 7.14% (p＜0.05) and 18.64% (p＜0.05) respectively in the liver; Compare with endoxan effect group, mouse is through the preventative filling stomach of 400mg/kg polysaccharide intraperitoneal injection of cyclophosphamide again, and SOD and GSH-Px activity improve 6.61% (p＜0.05) and 22.38% (p＜0.05) respectively in the liver; Compared with the control, irritate active 7.49% (p＜0.05) of improving of polysaccharide group liver SOD of stomach 400mg/kg separately.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (8)
1, a kind of enterobacter cloacae is characterized in that: this enterobacter cloacae is enterobacter cloacae Z0206CGMCC NO:2279.
2, a kind of preparation method of enterobacter cloacae Z0206 polysaccharide, its feature may further comprise the steps successively:
(1) enterobacter cloacae Z0206 actication of culture: the Z0206 of the enterobacter cloacae according to claim 1 bacterial classification that takes out freezing preservation, after room temperature is placed 1h, being inoculated into sterilization PDA adds on the rich solid medium, cultivate 36h～48h for 28 ℃～32 ℃, being transferred to PDA once more adds on the rich solid medium, cultivate 36h～48h, obtain activated spawn for 28 ℃～32 ℃;
(2) will activate good bacterial classification, being seeded to PDA adds and carries out shake flask fermentation in the liquid-rich substratum, 28 ℃～32 ℃ reciprocating vibrations are cultivated, incubation time 18h～20h obtains the ferment tank seed liquor, adds 70% fermention medium in the fermentor tank, 121 ℃ of steam sterilizing 20min, the inoculation seed liquor, fermentation time 48h～72h obtains containing the fermented liquid of enterobacter cloacae Z0206 polysaccharide;
(3) fermented liquid is concentrated into 1/10 of original volume in 60 ℃～70 ℃, 80 ℃ again～90 ℃ water-bath 1h～1.5h, centrifugal 10min～the 15min of 5000rpm collects supernatant liquor, adds 95% ethanol of 3～5 times of volume precoolings, 4 ℃ leave standstill 12h after, centrifugal 10min～the 15min of 5000rpm, precipitation is more successively with dehydrated alcohol, acetone and anhydrous diethyl ether washing, and is centrifugal, precipitation vacuum-drying promptly obtains the Crude polysaccharides powder;
(4) protease treatment: the Crude polysaccharides that step (3) is obtained is dissolved in the distilled water of heat of 20 times of volumes, uses Na
2CO
3Transfer pH to 7.0～9.0, add 0.25%～1% trypsin w/v), behind 55 ℃ of hydrolysis 1～2h, transfer pH to 5.0～5.7 with oxalic acid, add 0.25%～1% papoid (w/v) then, behind 65 ℃～70 ℃ hydrolysis 2h～3h, in 100 ℃ of heating in water bath 5～6min, to stop enzyme reaction;
(5) trichloroacetic acid method deproteinated: get the protease treatment liquid that obtains after the step (4), with oxalic acid adjust pH to 7.0, add 2%～4% trichoroacetic acid(TCA) (w/v), after the room temperature lower magnetic force stirred 1h, the centrifugal 10～15min of 11000rpm got supernatant;
(6) Sevag method deproteinated: add the Sevag reagent of 1/3 volume in the supernatant liquor that step (5) obtains, fully vibration mixes 20～30min, the sufficient standing layering, and the centrifugal 5min of 4000rpm, albumen precipitation no longer appears in repetitive operation several to two-phase interface; Use 95% alcohol chromatography of 3～5 times of volume precoolings then, the precipitation of gained is used dehydrated alcohol, acetone and anhydrous diethyl ether washed twice more successively, and vacuum-drying obtains the deproteinated polysaccharide.
(7) decolouring: will be made into 0.5% polysaccharide soln through the polysaccharide behind step (6) deproteinated, and transfer pH to 8.0, and under 50 ℃, drip 20% H with ammoniacal liquor
2O
2, be faint yellow to solution, insulation 2h is neutralized to 7.0 with dilute hydrochloric acid then; Tap water dialysis 2～3d behind redistilled water dialysis 2～3d, adds 95% ethanol of 3～5 times of volume precoolings, 4 ℃ leave standstill 12h after, the centrifugal 10min～15min of 5000rpm, precipitation vacuum-drying, polysaccharide products.
3, the preparation method of enterobacter cloacae Z0206 polysaccharide according to claim 2 is characterized in that: in described step (1) and (2), the prescription of PDA enriched medium is: potato 20%, peptone 0.2～0.5%, yeast extract 0.3%, glucose 2%, agar powder 1.6%; The prescription of fermention medium is: sucrose 2.5%, yeast extract 0.5%, peptone 0.5%, K
2HPO
40.2%, KH
2PO
40.1%, MgSO40.05%.
4, the preparation method of enterobacter cloacae Z0206 polysaccharide according to claim 2 is characterized in that: in the described step (2), conditions of flask fermentation is: inoculum size 3%, reciprocating vibration stroke 4～10cm, oscillation frequency 200～250rpm.
5, the preparation method of enterobacter cloacae Z0206 polysaccharide according to claim 2, it is characterized in that: in the described step (2), the ferment tank condition is: inoculum size 3%, pH is 7.0,30 ℃ of temperature, air flow is 0.25～0.75vvm, and mechanical stirring rotating speed 200～300rpm, every 12h replenish 1%～3% glucose to fermentor tank.
6, the preparation method of enterobacter cloacae Z0206 polysaccharide according to claim 2 is characterized in that: the prescription of the Sevag reagent in the described step (6) is that the volume ratio of chloroform and propyl carbinol is 4:1.
7, a kind of as enterobacter cloacae Z0206 polysaccharide as described in the claim 2 in preparation enhancing body anti-oxidant function and the healthcare products of immunoloregulation function or the application in the additive.
8, a kind of as the application of enterobacter cloacae Z0206 polysaccharide as described in the claim 2 in preparation antitumor drug and antiviral.
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| CN106085896A (en) * | 2016-05-10 | 2016-11-09 | 清华大学深圳研究生院 | Enterobacter cloacae and application thereof |
| CN106085896B (en) * | 2016-05-10 | 2019-07-09 | 清华大学深圳研究生院 | Enterobacter cloacae and application thereof |
| CN111518710A (en) * | 2019-02-02 | 2020-08-11 | 常熟理工学院 | Enterobacter strain and application thereof in preparation of microbial polysaccharide |
| CN111518710B (en) * | 2019-02-02 | 2022-03-29 | 常熟理工学院 | Enterobacter strain and application thereof in preparation of microbial polysaccharide |
| CN113186131A (en) * | 2021-04-30 | 2021-07-30 | 广州绿曦生物科技有限公司 | Alga-lysing microbial agent and application thereof |
| CN113186131B (en) * | 2021-04-30 | 2023-10-27 | 广州绿曦生物科技有限公司 | Algicidal microbial agent and application thereof |
| CN114292343A (en) * | 2021-12-31 | 2022-04-08 | 华南师范大学 | Preparation method of perennial cerasus extracellular polysaccharide and intracellular polysaccharide and application of perennial cerasus extracellular polysaccharide and intracellular polysaccharide in regulating intestinal microbial flora and reducing blood sugar |
| CN114292343B (en) * | 2021-12-31 | 2022-11-04 | 华南师范大学 | Preparation method of perennial cerasus extracellular polysaccharide and intracellular polysaccharide and application of perennial cerasus extracellular polysaccharide and intracellular polysaccharide in regulating intestinal microbial flora and reducing blood sugar |
| CN114437985A (en) * | 2022-02-18 | 2022-05-06 | 南京工业大学 | Enterobacter aerogenes and application thereof in synthesizing microbial polysaccharide |
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