CN1530439A - Hair weeds bacterial culture and culturing product - Google Patents
Hair weeds bacterial culture and culturing product Download PDFInfo
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- CN1530439A CN1530439A CNA031191010A CN03119101A CN1530439A CN 1530439 A CN1530439 A CN 1530439A CN A031191010 A CNA031191010 A CN A031191010A CN 03119101 A CN03119101 A CN 03119101A CN 1530439 A CN1530439 A CN 1530439A
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- hair weeds
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Abstract
A cell culture process for flagelliforme includes such steps as separating the cells from flagelliforme frond to obtain high-activity flagelliforme cells, culturing them in culture medium to obtain its seed cells, storing them at 8-12 deg.C under 500 lux or freeze drying for long-term storage, large-scale culturing to obtain the cultured product, and extracting flagelliforme polyol.
Description
Technical field
The present invention relates to the production technology that a kind of cell culture technology is particularly related to a kind of hair weeds cells culture technique and the polysaccharide of delivering vegetables.
Background technology
Deliver vegetables (Nostoc flagelliforme) be a kind of land natural disposition blue-green algae, be rich in polysaccharide, amino acid and trace element, be subjected to liking of people as a kind of famous and precious food for a long time.In recent years, the pharmaceutical use of delivering vegetables comes into one's own day by day, but contained polysaccharide material enhancing body immunizing power in delivering vegetables, and the hot-water extraction of delivering vegetables thing the experiment proved that to have anti-tumor activity.From deliver vegetables, extract a kind of polysaccharide Nostoflan, through experiment confirm virus such as herpes simplex types 1 virus, human cytomegalic inclusion disease virus and the influenza virus etc. of some tool big envelopes are had antiviral activity, and can kill the tumour cell of isolated culture.Having developed to deliver vegetables abroad is the healthcare products of main raw material, and market is in very great demand.
The polysaccharide of delivering vegetables at present is to extract from the frond of delivering vegetables.Production and some functional foodstuffs, medicine production and the exploitation of polysaccharide of delivering vegetables all needs a large amount of delivering vegetables as raw material.Delivering vegetables so far can not artificial growth production, and the special ecotope of delivering vegetables requires and regional distribution, mainly is grown in desert---the semi-desert grassland area, and it is very slow to grow, and natural standing stock are quite limited.Gathering in a large number without limit to delivering vegetables has made the hair-like seaweed resource subject to severe risks of damage, so that is on the verge of exhaustion.Ningxia is China one of main product ground of delivering vegetables, according to investigations, about 360.7 ten thousand hectares of the area of delivering vegetables the sixties 20th century of Ningxia vegetatively, reduce to 173.3 ten thousand hectares to the mid-80, do not reduced to 1,000,000 hectares of less thaies to the nineties in 20th century, the output of delivering vegetables then drops to about 0.1 kilogram of present per hectare from the per hectare 3-7.5 kilogram of the seventies.Unreasonable the gathering of delivering vegetables also done great damage to the vegetation of delivering vegetables vegetatively, cause desertification of land, ecotope goes from bad to worse.In view of this situation, the Chinese government completely forbids gathering, process and selling of delivering vegetables from July, 2000, therefore the production that to deliver vegetables is raw material can't be carried out, deliver vegetables polysaccharide as a kind of biologically active substance with applications well prospect, and its exploitation and production is not because of there being raw material sources to be difficult to carry out.
Summary of the invention
The present invention be directed to the research that the problems referred to above are carried out, its objective is and change the present situation that the polysaccharide of delivering vegetables can only extract from the frond of delivering vegetables, utilize cell culture technology, separate and obtain hair weeds cells, hair weeds cells is cultivated, from culture, nutrient solution, extract deliver vegetables polysaccharide or direct raw material, fundamentally solved the restriction that the raw material problem is used the polysaccharide of delivering vegetables as functional foodstuff, drug manufacture.
Technical scheme of the present invention is: with the hair weeds cells is cultivated material, and step is as follows:
(1) gets a little and deliver vegetables frond after the surface sterilization flushing, therefrom carry out cellular segregation, the hair weeds cells that obtains having vigorous vitality; (2) hair weeds cells is inserted in the substratum, obtain the hair weeds cells kind through cultivation, the cell kind is put 8-12 ℃, preservation under the low light level, and transfer once January, or adopt the long-term preservation of lyophil preservation method; (3) spread cultivation with the hair weeds cells kind, insert in the substratum again and cultivate, obtain cell culture through collection.
The cell culture that the present invention obtains, nutrient solution can directly extract deliver vegetables polysaccharide or direct raw material as functional foodstuff, drug manufacture.
It is as follows that hair weeds cells is cultivated concrete grammar:
(1) separates hair weeds cells: take by weighing and deliver vegetables on a small quantity in triangular flask, after the surface sterilization flushing, carry out cellular segregation with enzymolysis process or mechanical phonograph recorder separation.Enzymolysis process: aseptic condition adds down the macerozyme solution 30mL of filtration sterilization, and rotating speed 120r/min vibration 1-2 hour adds granulated glass sphere after changing enzyme liquid for the last time, and cell is disperseed, after filtering, and the centrifugal collection hair weeds cells of filtered liquid; Mechanical phonograph recorder separation: add an amount of physiological saline or substratum, fully grind, filter the centrifugal collection hair weeds cells of filtered liquid.
(2) cultivate the hair weeds cells kind: a small amount of hair weeds cells is inserted in the substratum pH8.5-9.0, temperature 24-32 ℃, time obtains the hair weeds cells kind for cultivation in 15-18 days, and the cell kind is put 8-12 ℃, preservation under the low light level, transfer once January, or adopt the long-term preservation of lyophil preservation method;
(3)---cultivation---culture spreads cultivation: after the hair weeds cells kind of a small amount of preservation is spread cultivation, inserts in the substratum, and pH8.5-9.0, culture temperature is 24-32 ℃, the illumination cultivation time is 10-15 days.Medium centrifugal is abandoned supernatant liquor after handling, and gets cell culture.
It is as follows to extract the polysaccharide concrete grammar of delivering vegetables the cell culture that obtains from the present invention, the nutrient solution:
Culture adds water, and is centrifugal behind the boiling water bath, or medium centrifugal, and supernatant concentration adds ethanol, precipitate centrifugal, the throw out absolute ethanol washing, vacuum-drying, polysaccharide or direct raw material as food, healthcare products or drug manufacture obtain delivering vegetables.
Advantage of the present invention and positively effect are: the polysaccharide because the production of employing cell culture method is delivered vegetables has changed the present situation that can only extract polysaccharide from the frond of delivering vegetables; The preservation for a long time of hair weeds cells kind is directly used in production through enlarged culturing, does not consume wild hair-like seaweed resource, does not destroy the ecotope vegetatively of delivering vegetables; Substratum is formed and the control culture condition by adjusting, and can change cell culture and become to be grouped into, and makes some specific cells product obtain accumulation; Cell culture, nutrient solution can extract deliver vegetables polysaccharide or direct raw material as food, healthcare products or drug manufacture.Solved because of what forbid delivering vegetables and gathered, process and sell, and the production of delivering vegetables to raw material can't be carried out, thereby limited problem that the polysaccharide of delivering vegetables is restricted as a kind of its research for application and development of biologically active substance with applications well prospect etc.Save the natural resources of delivering vegetables, made hair-like seaweed resource avoid destroying, protected ecotope.
Embodiment
Embodiment 1:
(1) separate hair weeds cells:
Take by weighing 1g and deliver vegetables in triangular flask, surface sterilization, aseptic water washing.Aseptic condition adds the macerozyme solution 30mL of filtration sterilization down, contains polygalacturonase 0.5% in the enzyme solution, N.F,USP MANNITOL 0.8%, T 500 potassium 1%.Triangular flask places on the shaking table, rotating speed 120r/min, and stroke 4-5cm, 25 ℃ of temperature, 2 hours time, every therebetween 30min changes an enzyme liquid, changes the granulated glass sphere that adds diameter 2mm in the enzyme liquid Vee formation bottle for the third time.Enzymolysis solution is with 800 order metal mesh filters after two hours, and centrifugal 15 minutes of filtered liquid 3000r/min collects hair weeds cells.
(2) cultivate the hair weeds cells kind:
After hair weeds cells washes 3 times with substratum, insert in the substratum, substratum is formed: sodium bicarbonate 1.2%, SODIUMNITRATE 0.12%, ammonium nitrate 0.03%, add dipotassium hydrogen phosphate, calcium chloride, sal epsom, copper sulfate, ferrous sulfate, zinc sulfate, Sodium orthomolybdate, boric acid, cobalt chloride, pH8.5,24 ℃, the 4000lux light intensity leaves standstill cultivated 10 days, Light To Dark Ratio 12: 12 gets the hair weeds cells kind.The cell kind is put 8 ℃, preservation under the 500lux low light level, and transfer once January, or adopt the long-term preservation of lyophil preservation method.
(3)---cultivation---culture spreads cultivation:
Earlier the hair weeds cells kind is spread cultivation, insert in the 15L air lift type photoreactor with 15% inoculum size then, the composition of its substratum is: sodium bicarbonate 1.2%, SODIUMNITRATE 0.18% is added dipotassium hydrogen phosphate, calcium chloride, sal epsom, pH8.5, culture temperature is 28 ℃, illumination cultivation 10 days.Centrifugal 30 minutes of nutrient solution 4000r/min abandons supernatant liquor, gets the hair weeds cells culture.
Embodiment 2:
Take by weighing 1g and deliver vegetables, surface sterilization is placed in the mortar, adds about 30mL stroke-physiological saline solution or substratum, and aseptic condition grinds down fully, 800 order metal mesh filters, filtered liquid is centrifugal, collects hair weeds cells, cultivate the hair weeds cells kind.The cell kind is put 10 ℃ of preservations, or in the back access substratum that spreads cultivation, inoculum size 20%, pH9, culture temperature is 30 ℃, illumination cultivation 15 days, all the other are with embodiment 1.
Embodiment 3:
The hair weeds cells kind is put 12 ℃ of preservations.The cell kind spreads cultivation the back with in the 15% inoculum size access reactor, and substratum is formed: sodium bicarbonate 1.6%, SODIUMNITRATE 0.12%, ammonium nitrate 0.03%, add dipotassium hydrogen phosphate, calcium chloride, sal epsom, pH8.75,32 ℃, the 4500lux light intensity, Light To Dark Ratio 16: 8, illumination cultivation 12 days.All the other are with embodiment 1 or embodiment 2.
Embodiment 4:
The hair weeds cells kind spreads cultivation the back with in the 15% inoculum size access reactor, and substratum is formed: sodium bicarbonate 1.6%, add dipotassium hydrogen phosphate, calcium chloride, sal epsom, SODIUMNITRATE, ferrous sulfate.Illumination cultivation 12 days.All the other are with embodiment 1 or embodiment 2.
Embodiment 5:
The extraction polysaccharide of delivering vegetables: culture adds water, put in the boiling water bath 1 hour, centrifugal 30 minutes of 3000r/min, supernatant liquor concentrates 4 times for 80 ℃, 95% ethanol that adds 2 times of volumes, precipitation 8h, centrifugal 15 minutes of 4000r/min, throw out absolute ethanol washing, 55 ℃, 750mmHg vacuum-drying obtains the polysaccharide of delivering vegetables, and all the other are with embodiment 1 or 2 or 3 or 4.
Embodiment 6:
Centrifugal 30 minutes of nutrient solution 3000r/min gets supernatant liquor, concentrates 4 times, adds 95% ethanol of 2 times of volumes, precipitate 8 hours, centrifugal 15 minutes of 4000r/min, throw out absolute ethanol washing, 55 ℃, the polysaccharide of delivering vegetables is extracted in 750mmHg vacuum-drying, and all the other are with embodiment 1 or 2 or 3 or 4.
Embodiment 7:
Nutrient solution film-type evaporator evaporation concentration, 60 ℃ in gained algae mud, 750mmHg vacuum-drying, the algae powder of must delivering vegetables is used to produce healthcare products or medicine.All the other are with embodiment 1 or 2 or 3 or 4.
Claims (5)
1, a kind of hair weeds cells is cultivated, and it is characterized in that with the hair weeds cells being cultivated material, specifically comprises the steps:
(1) gets a little and deliver vegetables after the surface sterilization flushing, therefrom carry out cellular segregation, the hair weeds cells that obtains having vigorous vitality;
(2) hair weeds cells is inserted in the substratum, obtain the hair weeds cells kind through cultivation, the cell kind is put 8-12 ℃, preservation under the low light level, and transfer once January, or adopt the long-term preservation of lyophil preservation method;
(3) the hair weeds cells kind inserts in the substratum and cultivates through spreading cultivation, and obtains cell culture through collection;
2, hair weeds cells according to claim 1 is cultivated, and it is characterized in that carrying out from the frond of delivering vegetables the hair weeds cells that cellular segregation obtains is with enzymolysis process or mechanical phonograph recorder separation.
3, hair weeds cells according to claim 1 and 2 is cultivated, it is characterized in that the hair weeds cells that separation is obtained inserts in the substratum, its substratum is carbon source with carbonate, with one or both nitrate or ammonium salt is nitrogenous source, add in phosphoric acid salt, sylvite, vitriol, magnesium salts, molysite, Sodium orthomolybdate, boric acid, calcium chloride, the cobalt chloride partly or entirely; Culture temperature is 24-32 ℃, 10-15 days illumination cultivation time.
4, hair weeds cells according to claim 1 and 2 is cultivated, and after it is characterized in that the hair weeds cells kind spread cultivation, inserts and adopts photoreactor that hair weeds cells is cultivated in the substratum; With carbonate or carbonic acid gas is carbon source, does not add nitrogenous source or is nitrogenous source with one or both nitrate and ammonium salt, adds mineral element.
5,, it is characterized in that from cell culture, nutrient solution, directly extracting deliver vegetables polysaccharide or direct raw material as food, healthcare products or drug manufacture according to the resulting hair weeds cells cultured products of arbitrary claim among the claim 1-4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03119101 CN1244690C (en) | 2003-03-14 | 2003-03-14 | Hair weeds bacterial culture and culturing product |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03119101 CN1244690C (en) | 2003-03-14 | 2003-03-14 | Hair weeds bacterial culture and culturing product |
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| CN1530439A true CN1530439A (en) | 2004-09-22 |
| CN1244690C CN1244690C (en) | 2006-03-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 03119101 Expired - Fee Related CN1244690C (en) | 2003-03-14 | 2003-03-14 | Hair weeds bacterial culture and culturing product |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007259738A (en) * | 2006-03-28 | 2007-10-11 | Tianjin Science & Technology Univ | Method for carrying out culture of nostoc flagelliforme cell and extraction of polysaccharide of nostoc flagelliforme by linking to each other |
| CN100427581C (en) * | 2005-12-01 | 2008-10-22 | 天津科技大学 | Process for solid culture of hair weeds cells |
| CN101864470A (en) * | 2010-06-11 | 2010-10-20 | 山东轻工业学院 | Method for increasing yield of extracellular polysaccharide produced by submerged fermentation of long thread moss cells |
| CN102669520A (en) * | 2012-05-07 | 2012-09-19 | 天津科技大学 | Nostoc flagelliforme polysaccharide oral liquid and preparation method thereof |
| CN103173389A (en) * | 2013-03-15 | 2013-06-26 | 博赢（昆山）生物科技有限公司 | Method for culturing hair weed cells industrially |
| CN103463121A (en) * | 2013-08-15 | 2013-12-25 | 天津科技大学 | Application of nostoc flagelliforme polysaccharides used for preparation of immunomodulatory drugs or health products |
| CN104845908A (en) * | 2015-04-28 | 2015-08-19 | 河南科技大学 | Strontium-rich hair weed cell and culture method thereof |
| CN110169984A (en) * | 2019-05-23 | 2019-08-27 | 天津科技大学 | A kind of application of the algae powder in adjusting intestinal microflora of delivering vegetables |
-
2003
- 2003-03-14 CN CN 03119101 patent/CN1244690C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427581C (en) * | 2005-12-01 | 2008-10-22 | 天津科技大学 | Process for solid culture of hair weeds cells |
| JP2007259738A (en) * | 2006-03-28 | 2007-10-11 | Tianjin Science & Technology Univ | Method for carrying out culture of nostoc flagelliforme cell and extraction of polysaccharide of nostoc flagelliforme by linking to each other |
| CN101864470A (en) * | 2010-06-11 | 2010-10-20 | 山东轻工业学院 | Method for increasing yield of extracellular polysaccharide produced by submerged fermentation of long thread moss cells |
| CN101864470B (en) * | 2010-06-11 | 2013-01-09 | 山东轻工业学院 | Method for increasing yield of extracellular polysaccharide produced by submerged fermentation of long thread moss cells |
| CN102669520A (en) * | 2012-05-07 | 2012-09-19 | 天津科技大学 | Nostoc flagelliforme polysaccharide oral liquid and preparation method thereof |
| CN103173389A (en) * | 2013-03-15 | 2013-06-26 | 博赢（昆山）生物科技有限公司 | Method for culturing hair weed cells industrially |
| CN103463121A (en) * | 2013-08-15 | 2013-12-25 | 天津科技大学 | Application of nostoc flagelliforme polysaccharides used for preparation of immunomodulatory drugs or health products |
| CN104845908A (en) * | 2015-04-28 | 2015-08-19 | 河南科技大学 | Strontium-rich hair weed cell and culture method thereof |
| CN104845908B (en) * | 2015-04-28 | 2018-08-31 | 河南科技大学 | A kind of richness strontium hair weeds cells and its cultural method |
| CN110169984A (en) * | 2019-05-23 | 2019-08-27 | 天津科技大学 | A kind of application of the algae powder in adjusting intestinal microflora of delivering vegetables |
| CN110169984B (en) * | 2019-05-23 | 2023-04-07 | 天津科技大学 | Application of Nostoc flagelliforme powder in regulating intestinal flora structure |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1244690C (en) | 2006-03-08 |
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