CN101475916A - Enterobacter cloacae, selenium-rich polysaccharide thereof and use of selenium-rich polysaccharide - Google Patents
Enterobacter cloacae, selenium-rich polysaccharide thereof and use of selenium-rich polysaccharide Download PDFInfo
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Abstract
The invention discloses an Enterobacter cloacae Z0206, which has been stored in 'China microorganism strain preservation management committee common micro organism center' at December 3rd, 2007 and whose storage number is CGMCC NO 2279; also discloses is an Enterobacter cloacae Selenium-rich polysaccharide preparation method and applications of Selenium-rich polysaccharide. The invention has advantages that: the invention has simple production process of extracting the Selenium-rich polysaccharide, high product yield and higher purity, higher Selenium-rich levels; the selenium-enriched polysaccharide prepared by the method can significantly improve the lymphopoiesis capability as well as cytokine level of mice with low immune capacity, and has significant role in the regulation of the immune function of mice with low immune capacity; the selenium-enriched polysaccharide prepared by the method can significantly improve the activity of anti-oxidase, to enhance the function of the antioxidant defense system, has a clear protective effect to the oxidative damage of RAW264.7 cells induced by H2O2, which is an effective antioxidant.
Description
Technical field
The present invention relates to microbiology and technical field of bioengineering, a kind of research of passing through the anti-selenium domestication of bacterium, the research of rich selenium and rich selenium polysaccharide extraction purge process of design especially, preparation and application have the method for the rich selenium polysaccharide of enterobacter cloacae Z0206 bacterial activity of anti-oxidant and immunoloregulation function.
Background technology
Selenium more and more is subjected to the attention of scientific circles as a kind of important trace element.Studies show that selenium has enhance immunity power, anti-oxidant, detoxifcation, suppresses functions such as tumour, preventing cardiovascular disease generation.Inorganic selenium toxicity is far above organic selenium compounds, and bioavailability is low.Have only inorganic selenium is converted into organoselenium, just have edible and health care value.The bio-organic of selenium mainly is to produce organoselenium by animal, plant, microbial transformation inorganic selenium.Carrying out organising not only of selenium by biology can be for the mankind provides food and the healthcare products that are rich in organoselenium, and can be a method that has bright prospects for livestock industry production provides the additive that is rich in organoselenium again.
The polysaccharide that part can be carried out the microorganism secretion that selenium organises has immune-enhancing activity, show mediation and regulating effect to host immune, maturation, differentiation and propagation by the 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, these polysaccharide show 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 pass through repeatedly screening, purifying, separate having obtained a strain exocellular polysaccharide high yield bacterial isolates enterobacter cloacae Z0206 from Ganoderma (Shaanxi).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 ".Preliminary study shows, bacterial strain enterobacter cloacae Z0206 has higher tolerance to selenium, growth can be converted into organoselenium with inorganic selenium on the inorganic selenium substratum, combine with its excretory polysaccharide, arrive outside the born of the same parents with the excretory form, have the high characteristics of output, and the rich selenium polysaccharide of this bacterial activity also has certain anti-oxidant and immunoregulatory activity.The rich selenium polysaccharide of this bacterial activity has higher actual application value in biological health-care product and Additive Production and exploitation.Rich selenium by bacterium is cultivated, and the rich selenium polysaccharide of production bacterial activity meets the demand for development of healthcare products exploitation and green feed additive as the natural health biotechnological formulation, has important social reality meaning and ecological significance.
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.
The preparation method of the rich selenium polysaccharide of a kind of enterobacter cloacae Z0206 may further comprise the steps successively:
(1) the anti-selenium ability domestication of enterobacter cloacae Z0206: picking enterobacter cloacae Z0206 bacterial classification, successively coating selenium content is on the PDA enriched medium flat board of 10,20,30,40,50 μ g/mL, carries out the domestication of selenium concentration gradient.Each gradient is cultivated 24～36h in 28～32 ℃.The well-grown bacterium colony of picking separates in the flat board line from the PDA enriched medium flat board of selenium content 50 μ g/mL at last, and the purifying back bevel is preserved.
(2) the rich selenium research of enterobacter cloacae Z0206:, determine that the best adds selenium time and incubation time by measuring the growth curve of bacterium.
Bacterial classification from the inclined-plane after the anti-selenium domestication of picking is inoculated in selenium content and is respectively on the PDA enriched medium of 10,15,20,25,30,35 μ g/mL, cultivates 24-48h for 28～32 ℃, observes the thalline colour-change and determines best selenium concentration.For avoiding too much inorganic selenium to be converted into simple substance selenium, improve the organoselenium transformation efficiency, choose on the substratum slightly red, the well-grown selenium concentration of thalline color as best selenium concentration.
Bacterial classification from the inclined-plane after the anti-selenium domestication of picking behind PDA culture medium flat plate activation culture 24～36h, is inoculated in the triangular flask that the PDA substratum is housed.28～32 ℃ of shaking culture every the aseptic taking-up of 1h 1mL nutrient solution, detect the absorbance at 580nm place with spectrophotometer.Draw the growth curve of bacterium.Determine to add selenium time and incubation time by growth curve.
(3) with the enterobacter cloacae Z0206 bacterial classification after the anti-selenium domestication, 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, obtain the ferment tank seed liquor, add 70% fermention medium in the fermentor tank, 121 ℃ of steam sterilizing 20min, the inoculation seed liquor, add aseptic sodium selenite solution when cultivating 13h, making the final selenium content of substratum is 25 μ g/mL, and fermentation time 48h～72h obtains containing the fermented liquid of the rich selenium polysaccharide of enterobacter cloacae Z0206;
(4) 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 rich selenium Crude polysaccharides powder;
(5) protease treatment: the rich selenium Crude polysaccharides that step (4) is obtained is dissolved in the distilled water of heat of 20 times of volumes, transfer pH to 7.0～9.0 with Na2CO3, 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;
(6) trichloroacetic acid method deproteinated: get the protease treatment liquid that obtains after the step (5), 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;
(7) Sevag method deproteinated: add the Sevag reagent of 1/3 volume in the supernatant liquor that step (6) 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 rich selenium polysaccharide of deproteinated;
(8) decolouring: will be made into 0.5% solution through the rich selenium polysaccharide behind step (7) 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 distill 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, rich selenium polysaccharide elaboration.
In described step (1), (2) or (3), the prescription of PDA enriched medium is: potato 20%, peptone 0.2～0.5%, yeast extract 0.3%, glucose 2%, 1.6%, 121 ℃ of sterilization of agar powder 20min.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 (3), conditions of flask fermentation is: inoculum size 3～5 rings, reciprocating vibration stroke 4～10cm, oscillation frequency 200～250rpm.
In the described step (3), 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.
The rich selenium polysaccharide of a kind of enterobacter cloacae Z0206 is in preparation enhancing body anti-oxidant function and the healthcare products of immunoloregulation function or the application in the additive.
The application of the rich selenium polysaccharide of a kind of enterobacter cloacae Z0206 in preparation antitumor drug and antiviral.
Advantage of the present invention:
(1) adopts polysaccharide, protein and selenium content in phenolsulfuric acid method, Kjeldahl determination, the rich selenium polysaccharide of fluorescence spectrophotometry enterobacter cloacae Z0206.Measurement result shows: polysaccharide, protein and selenium content are respectively in the rich selenium Crude polysaccharides: 69.40%, 25.68% and 65.70mg/kg; Polysaccharide, protein and selenium content are respectively in the rich selenium polysaccharide elaboration: 99.97%, 0.03% and 35.56mg/kg.The result shows that the production process of the rich selenium polysaccharide of said extracted is simple, and product production and purity are higher, and rich selenium degree is higher.
(2) the rich selenium polysaccharide of enterobacter cloacae Z0206 by present method preparation can significantly improve immunocompromised mouse lymphocyte multiplication capacity and cytokine levels, and the immunocompromised immune function of mice is had significant regulating effect.
(3) the rich selenium polysaccharide of enterobacter cloacae Z0206 by present method preparation can significantly improve activities of antioxidant enzymes, strengthens the function of anti-oxidative defense system, to H
2O
2The oxidative damage of the RAW264.7 cell that causes has significant protective effect, is a kind of good antioxidant.
Description of drawings
Fig. 1 is enterobacter cloacae Z0206 growth curve in the embodiment of the invention.
Embodiment
Below in conjunction with specific examples technical scheme of the present invention is described further:
The preparation method of the rich selenium polysaccharide of embodiment 1, enterobacter cloacae Z0206
(1) the anti-selenium ability domestication of enterobacter cloacae Z0206: PDA enriched medium prescription is as follows: potato 20%, peptone 0.3%, yeast extract 0.3%, glucose 2%, agar powder 1.6%.Picking enterobacter cloacae Z0206 bacterial classification, successively coating selenium content is on the PDA enriched medium flat board of 10,20,30,40,50 μ g/mL, carries out the domestication of selenium concentration gradient.Each gradient is cultivated 36h in 32 ℃.The well-grown bacterium colony of picking separates in the flat board line from the PDA enriched medium flat board of selenium content 50 μ g/mL at last, and the purifying back bevel is preserved.
(2) the rich selenium research of enterobacter cloacae Z0206:
The bacterial classification of anti-selenium that picking has been tamed from the inclined-plane is inoculated in selenium content and is respectively on the PDA enriched medium of 10,15,20,25,30,35 μ g/mL, cultivates 36h for 30 ℃, observes the thalline colour-change and determines best selenium concentration.The result shows that along with the increase of selenium concentration in the solid medium, thalline reddens gradually.30, redness is darker during 35 μ g/ml, slightly take on a red color during 25 μ g/ml and bacteria growing better.For avoiding too much inorganic selenium to be converted into simple substance selenium, improve the organoselenium transformation efficiency, determine that 25 μ g/ml are best selenium concentration.
By measuring the growth curve of the bacterial strain of anti-selenium, determine that the best adds selenium time and incubation time.The bacterial strain of anti-selenium that picking is tamed from the inclined-plane behind the PDA culture medium flat plate activation culture 12h, is inoculated in the triangular flask that the 100mLPDA substratum is housed.Be positioned over 30 ℃ of shaking culture in the shaking table, therefrom take out the 1mL nutrient solution at sterilisable chamber, with the light absorption value under the spectrophotometric determination 580nm every 1h.Determine the growth curve (Fig. 1) of anti-selenium bacterium.
With reference to figure 1: enterobacter cloacae Z0206 is in logarithmic phase at 4h-15h, and 15h-24h is in stationary phase.The logarithmic phase bacterial metabolism is vigorous, the transformation efficiency height, and it is early stronger more to the movable inhibition of bacterial metabolism to add the selenium time, considers that from transformation efficiency and production cycle select the 13rd hour for the best adds the selenium time, best incubation time is 48h.
(3) the rich selenium polysaccharide preparation of fermentation liquid of enterobacter cloacae Z0206: in the 250ml triangular flask, add 70mlPDA and add the liquid-rich substratum, 121 ℃ of sterilization 20min, behind inoculation of medium 5 ring bacterial classifications, 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 are that pH7.0,30 ℃ of temperature, air flow are 0.5vvm, mechanical stirring rotating speed 200rpm, add aseptic sodium selenite solution when cultivating 13h, making the final selenium content of substratum is 25 μ g/mL, every 12h replenishes 1% glucose to fermentor tank, and fermentation time 48h obtains containing the fermented liquid of the rich selenium polysaccharide of enterobacter cloacae Z0206.
(4) separation and Extraction of the rich selenium polysaccharide of enterobacter cloacae Z0206:
The extraction of a. rich selenium 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 95% ethanol 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 dehydrated alcohol, acetone and the anhydrous diethyl ether of precooling more successively, centrifugal, at last precipitation is placed vacuum drier dry, obtain rich selenium Crude polysaccharides powder.
B. protease treatment:
Get rich selenium 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 rich selenium polysaccharide of deproteinated.
E. decolouring:
Rich selenium polysaccharide behind the deproteinated is made into 0.5% solution, transfers pH value to 8.0, 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 48h behind the distill water dialysis 48h, adds 95% ethanol of 3 times of volume precoolings, 4 ℃ leave standstill 12h after, the centrifugal 10min of 5000rpm, precipitation vacuum-drying, rich selenium polysaccharide elaboration.
Embodiment 2, application phenolsulfuric acid method detect polysaccharide content in the rich selenium polysaccharide of enterobacter cloacae Z0206
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 rich selenium 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 rich selenium polysaccharide of enterobacter cloacae Z0206.
Adopt the Kjeldahl determination (GB/T 5009.5-1985) of national Specification to measure protein content in the rich selenium polysaccharide of enterobacter cloacae Z0206.
Embodiment 4, the rich content of selenium in selenium polysaccharide of application fluorescence spectrophotometry enterobacter cloacae Z0206.
Adopt the spectrophotofluorimetry (GB12399-90) of national Specification to measure the rich content of selenium in selenium polysaccharide of enterobacter cloacae Z0206.
Embodiment 2, embodiment 3 and example 4: adopt polysaccharide, protein and selenium content in phenolsulfuric acid method, Kjeldahl determination, the rich selenium polysaccharide of fluorescence spectrophotometry enterobacter cloacae Z0206.Measurement result shows: polysaccharide, protein and selenium content are respectively in the rich selenium Crude polysaccharides: 69.40%, 25.68% and 65.70mg/kg; Polysaccharide, protein and selenium content are respectively in the rich selenium polysaccharide elaboration: 99.97%, 0.03% and 35.56mg/kg.The result shows that the production process of the rich selenium polysaccharide of said extracted is simple, and product production and purity are higher, and rich selenium degree is higher.
The rich selenium polysaccharide anti-oxidant activity test of embodiment 4, enterobacter cloacae Z0206
The RAW264.7 cell (can purchase in Shanghai cell biological institute) of recovery is inoculated in the DMEM substratum that contains 10% foetal calf serum, 100 υ/mL penicillin and 100mg/L Streptomycin sulphate (can available from Hyclone company), when treating that cell grows to logarithmic phase, cell concn is adjusted to 1 * 10
5After individual/mL concentration, be inoculated in 96 well culture plates, every hole 100 μ L put 37 ℃, 5%CO
2Thermostat container in hatch (after treating cell attachment) behind the 24h, carry out drug study, be divided into negative control (adding contains the DMEM nutrient solution of 10% foetal calf serum), positive control (contains the H that final concentration is 200 μ mol/L
2O
2) and the administration group of various dose, the administration group adds the rich selenium polysaccharide solution of 100 μ L/ hole different concns respectively, makes its final concentration be respectively 400,200,100 μ g/mL, respectively establishes 4 repeating holes, after placing 37 ℃, the thermostat container of 5%CO2 to hatch 24h, adding final concentration is the H of 200 μ mol/L
2O
2After continuing to cultivate 12h, carry out the detection of index of correlation.
1, in above-mentioned culture plate, adds MTT solution (5mg/ml) 10 μ L/ holes, continue to cultivate 4h, after cultivating end, take out culture plate, the careful suction abandoned substratum in the hole, washes 1 time with serum free medium, add 100 μ LDMSO (can available from U.S. Santa Cruz company), place 37 ℃, the thermostat container of 5%CO2 to hatch 10min, after purple crystal dissolves fully, detect 570nm absorbancy (A) value of respectively organizing cell with microplate reader.Experimental group cell survival rate=(experimental group A value-blank group A value)/(normal control group A value-blank group A value) * 100%.
2, adopt the content of kit measurement cell interior SOD, GSH-Px and MDA.
Table 1 is depicted as the rich selenium polysaccharide of enterobacter cloacae Z0206 to H
2O
2Induce the influence of oxidative damage RAW264.7 cell growth;
Table 2 is depicted as the rich selenium polysaccharide of enterobacter cloacae Z0206 to H
2O
2Induce the influence of SOD, GSH-Px activity and MDA content in the oxidative damage RAW264.7 cell;
Table 1
Annotate: same column subscript letter different table differential different significantly (p＜0.05).
Compare with control group, it is the H of 200 μ M that the RAW264.7 cell adds final concentration
2O
2Cultivated 12 hours, the survival rate of cell reduces by 50.65% (p＜0.05); H with 200 μ M
2O
2The effect group is compared, and with the rich selenium polysaccharide pre-treatment of 100 μ g/mL, 200 μ g/mL and 400 μ g/mL 24 hours, adds the H that final concentration is 200 μ M again
2O
2, continuing to cultivate 10 hours, cell survival rate has improved 7.32% (p＜0.05), 5.25% (p＜0.05) and 22.72% (p＜0.05) respectively.
Table 2
Annotate: same column subscript letter different table differential different significantly (p＜0.05).
Compare H with control group
2O
2Effect (200 μ M, 12 hours) group intracellular SOD of RAW264.7 and GSH-Px activity have reduced by 45.21% (p＜0.05) and 57.89% (p＜0.05) respectively separately, and MDA content has improved 144.64% (p＜0.05); H with 200 μ M
2O
2The effect group is compared, and has improved 28.95% (p＜0.05) and 47.56% (p＜0.05) respectively with rich intracellular SOD of selenium polysaccharide pretreated group of 400 μ g/mL and GSH-Px activity, and MDA content has reduced by 28.22% (p＜0.05).
The rich selenium polysaccharide immunoregulation effect test of embodiment 5, enterobacter cloacae Z0206
Get 40 of ICR mouse (can available from Medical College of Zhejiang Univ.'s Experimental Animal Center) (male and female half and half), be divided into control group (I) at random, endoxan group (II), endoxan+rich selenium polysaccharide group (III) and rich selenium polysaccharide group (IV), I, II organizes and irritates stomach physiological saline 0.4mL every day, III, IV organizes and irritates the rich selenium polysaccharide 0.4mL (400mg/kg body weight) of stomach every day, II, the 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 0.2mL10% 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, blood sample collection prepares serum, adopts ELISA method kit measurement Cytokine of Serum IL-2 and TNF-alpha content.
2, gather spleen, adopt mtt assay to carry out the spleen lymphocyte proliferation test, measure 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, adjusting cell concn with the RPMI-1640 nutrient solution is 2 * 106/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, and 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 LDMSO 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.
Table 3 is the influence of the rich selenium polysaccharide of enterobacter cloacae Z0206 to IL-2 in the ICR mice serum and TNF-alpha levels;
Table 4 is the influence of the rich selenium polysaccharide of enterobacter cloacae Z0206 to ICR mouse spleen lymphocyte propagation.
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, IL-2 and TNF-alpha levels reduce by 41.04% (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 the rich selenium polysaccharide of 400mg/kg intraperitoneal injection of cyclophosphamide again, and IL-2 and TNF-alpha levels improve 15.24% (p＜0.05) and 50.57% (p＜0.05) respectively in the serum; Compared with the control, irritate in the rich selenium polysaccharide group serum of stomach 400mg/kg IL-2 and TNF-alpha levels improve 1.97% respectively (p〉0.05) and 20.49% (p＜0.05) separately.
Table 4
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 the rich selenium polysaccharide of 400mg/kg intraperitoneal injection of cyclophosphamide again, and ConA inductive splenic T lymphocytic proliferation rate and LPS inductive bone-marrow-derived lymphocyte proliferation rate improve 7.12% (p＜0.05) and 20.45% (p＜0.05) respectively; Compare with control group, the rich selenium polysaccharide group ConA inductive splenic T lymphocytic proliferation rate and the LPS inductive bone-marrow-derived lymphocyte proliferation rate of irritating stomach 400mg/kg separately improve 6.13% and 3.79% respectively, but difference is not remarkable.
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, the preparation method of the rich selenium polysaccharide of a kind of enterobacter cloacae Z0206, its feature may further comprise the steps successively:
(1) the anti-selenium ability domestication of enterobacter cloacae Z0206: picking enterobacter cloacae Z0206 bacterial classification, successively coating selenium content is on the PDA enriched medium flat board of 10,20,30,40,50 μ g/mL, carries out the domestication of selenium concentration gradient; Each gradient is cultivated 24～36h in 28～32 ℃; The well-grown bacterium colony of picking separates in the flat board line from the PDA enriched medium flat board of selenium content 50 μ g/mL at last, and the purifying back bevel is preserved;
(2) the rich selenium research of enterobacter cloacae Z0206:, determine that the best adds selenium time and incubation time by measuring the growth curve of bacterium;
Bacterial classification from the inclined-plane after the anti-selenium domestication of picking is inoculated in selenium content and is respectively on the PDA enriched medium of 10,15,20,25,30,35 μ g/mL, cultivates 24～48h for 28～32 ℃, observes the thalline colour-change and determines best selenium concentration; For avoiding too much inorganic selenium to be converted into simple substance selenium, improve the organoselenium transformation efficiency, choose on the substratum slightly red, the well-grown selenium concentration of thalline color as best selenium concentration;
Bacterial classification from the inclined-plane after the anti-selenium domestication of picking behind PDA culture medium flat plate activation culture 24～36h, is inoculated in the triangular flask that the PDA substratum is housed; 28～32 ℃ of shaking culture every the aseptic taking-up of 1h 1mL nutrient solution, detect the absorbance at 580nm place with spectrophotometer; Draw the growth curve of bacterium; Determine to add selenium time and incubation time by growth curve;
(3) with the enterobacter cloacae Z0206 bacterial classification after the anti-selenium domestication, 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, obtain the ferment tank seed liquor, add 70% fermention medium in the fermentor tank, 121 ℃ of steam sterilizing 20min, the inoculation seed liquor, add aseptic sodium selenite solution when cultivating 13h, making the final selenium content of substratum is 25 μ g/mL, and fermentation time 48h～72h obtains containing the fermented liquid of the rich selenium polysaccharide of enterobacter cloacae Z0206;
(4) 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 rich selenium Crude polysaccharides powder;
(5) protease treatment: the rich selenium Crude polysaccharides that step (4) 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;
(6) trichloroacetic acid method deproteinated: get the protease treatment liquid that obtains after the step (5), 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;
(7) Sevag method deproteinated: add the Sevag reagent of 1/3 volume in the supernatant liquor that step (6) 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 rich selenium polysaccharide of deproteinated;
(8) decolouring: will be made into 0.5% solution through the rich selenium polysaccharide behind step (7) 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 distill 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, rich selenium polysaccharide elaboration.
3, the preparation method of the rich selenium polysaccharide of enterobacter cloacae Z0206 according to claim 2, it is characterized in that: in described step (1), (2) or (3), 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%.
4, the preparation method of the rich selenium polysaccharide of enterobacter cloacae Z0206 according to claim 2, it is characterized in that: in the described step (2), conditions of flask fermentation is: inoculum size 3～5 rings, reciprocating vibration stroke 4～10cm, oscillation frequency 200～250rpm.
5, the preparation method of the rich selenium polysaccharide of enterobacter cloacae Z0206 according to claim 2, it is characterized in that: in the described step (2), the ferment tank condition is: inoculum size 3%, pH7.0,30 ℃ 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 the rich selenium polysaccharide of enterobacter cloacae Z0206 according to claim 2, it 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 the rich selenium polysaccharide of enterobacter cloacae Z0206 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 the rich selenium polysaccharide of enterobacter cloacae Z0206 as described in the claim 2 in preparation antitumor drug and antiviral.
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| CN106153664B (en) * | 2016-07-19 | 2018-05-08 | 大连工业大学 | A kind of method, test solution and method for preparing test solution for detecting marine polysaccharide antioxidation |
| CN112725230A (en) * | 2020-12-31 | 2021-04-30 | 广西壮族自治区农业科学院 | Selenium-resistant strain Enterobacter ludwigii GX-C3 and application thereof |
| CN112725230B (en) * | 2020-12-31 | 2022-04-08 | 广西壮族自治区农业科学院 | Selenium-resistant strain Enterobacter ludwigii GX-C3 and application thereof |
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