CN1392245A - Method for cultivating hair weeds cell - Google Patents

Abstract

The cultivation process of hair weel cell includes the steps of culture liquid compounding with sodium nitrate, dipotassium biphosphate, MgSO4.7H2O, CaCl2.2H2O, Na2SiO3.9H2O, micro amount of metal element solution PV and soil lixivium added into water successively; preparing hair weed cell homogenate with wild hair weed filament via washing, soaking in aseptic liquid, crushing in a homogeniser; aseptic purification of hair weed seed; culturing; real-time monitoring; collection and drying. The said process is simple, pollution-free, short in production period and high in production efficiency, and the product has comprehensive and high nutrients.

Description

A kind of method of cultivating hair weeds cells

Technical field

The present invention relates to biological technical field, more specifically relate to a kind of method of cultivating hair weeds cells.

Background technology

The beads algae is the algae monoid that class distribution region is wide, ecotope is complicated, morphological structure is various that Cyanophyta Cyanophyta Cyanophyceae Cyanophyceae section is grown Cutleriales Hormogonales nostocaceae Nostocaceae, gluey or the leather shape of its plant materials, the maturation plant body has multiple shapes such as sphere, lobate, thread, blister, hollow or solid, seek floating or give birth to growth.The beads algae is the main composition kind in many extreme environments, shine the trace that to find the beads algae in the extreme environments such as intensity, high salinity at high temperature, severe cold, strong ultraviolet radiation, utmost point low light, some kinds wherein as deliver vegetables (Nostoc flagelliforme), Nostoc commune (Nostoccommune), Nostoc (Nostoc sphaeroides) is nutritious can be edible, and has certain pharmaceutical use.Deliver vegetables since ancient times, Nostoc, Nostoc commune be exactly the natural traditional food of the Chinese nation, wherein delivers vegetables to be subjected to human consumer's favor for a long time because of its abundant nutrition and lucky pronunciation (with " making a good deal of money " partials), in many ancient books, all be documented.Deliver vegetables have another name called flagelliform nostoc, the hair, formal name used at school is sent out shape beads algae, it is universal edible Lu Sheng blue-green algae, desert half-desert area western in China and the northwestward often can be seen, before its edible and medicinal history can be traced back to 3,000 years, in recent decades aspects such as its morphology, ecology, physiology and developmental biology there has been certain research.Deliver vegetables have dietotherapy, function such as health care, nourishing; find a good sale at home and abroad always; the contradiction of environmental degradation that present development of resources brought and grassland protection etc. is becoming increasingly acute; have research to claim digging to deliver vegetables for 1 kilogram and can destroy 3 hectares of grassland, environmental destruction and natural resources atrophy decline situation that visible over-drastic digging produces are extremely serious.Exist the contradiction that is difficult to be in harmonious proportion between great demand and limited resources amount, the only way of solution is to carry out artificial culture of Nostoc flagelliforme and intensive culture production.Owing to deliver vegetables growth distribution in desert half-desert and area, grassland, living environment has certain specificity, its growth conditions needed such as temperature, moisture, illumination, light dark period, nutrition etc. are strict, be difficult to artificial culture at present and go out the sophisticated filament of delivering vegetables, even hair weeds cells does not have the report of cultured continuously success so far yet.

Summary of the invention

The purpose of this invention is to provide a kind of method of cultivating hair weeds cells, in the hope of be difficult to obtain under the situation that artificial culture obtains artificial culture thing at filament with the nutritive ingredient of delivering vegetables substantially, this method is simple, easy to operate, culture possesses the nutritive ingredient of delivering vegetables, can make all kinds of health promoting beverage, alleviate the problem of the market requirement of delivering vegetables.

In order to achieve the above object, the present invention has taked following technical measures: what 1, cultivate at first consideration is the prescription of nutrient solution, the present invention relatively filters out the cultivation of optimum hair weeds cells from the nutrient solution of 8 suitable blue algae growths nutrient solution prescription is the HGZ nutrient solution, NaNO ₃490-500mg/L, K ₂HPO ₄35-40mg/L, MgSO ₄7H ₂O 70-75mg/L, CaCl ₂2H ₂O 35-36mg/L, Na ₂SiO ₄9H ₂O 55-60mg/L, PIV 3ml/L, soil extract 3ml/L.Below be the prescription of other 7 nutrient solutions: 1 ^#Nutrient solution: Ca (NO ₃) ₂0.2g/L, K ₂HPO ₄0.075g/L, MgSO ₄7H ₂O 0.1g/L, Na ₂CO ₃0.2g/L, 0.5%Na ₂MoO ₄4ml, 0.5%HBO ₃4ml, 1%FeC ₆H ₅O ₇5H ₂O 0.5ml, 1%C ₆H ₈O ₇H ₂O0.5ml/L; 2 ^#Nutrient solution: CuSO ₄5H ₅O 19.6mg/L, ZnSO ₄7H ₂O 44.0mg/L, CoCl ₂6H ₂O 20.0mg/L, MnCl ₂4H ₂O 36.0mg/L, Na ₂MoO ₄2H ₂O 12.6mg/L, H ₃BO ₃618.4mg/L; 3 ^#Nutrient solution: 1 ^#: 2 ^#=1: 1000; 4 ^#Nutrient solution: MgSO ₄7H ₂O 0.2g/L, K ₂HPO ₄0.5g/L, NaCl 0.2g/L, CaSO ₄2H ₂O 0.1g/L, FeCl ₃6H ₂O 0.005g/L, CaCO ₃1.0g/L; 5# nutrient solution: MgSO ₄7H ₂O 125mg/L, KNO ₃250mg/L, CaCl ₂27mg/L, NaOH 3.76mg/L, FeSO ₄7H ₂O 1.0mg/L, A ₅1.0ml; 6 ^#Nutrient solution: Ca (NO ₃) ₄4H ₂O 0.04g/L, K ₂HPO ₄0.01g/L, MgSO ₄7H ₂O 0.025g/L, Na ₂CO ₃0.02g/L, Na ₂SiO ₃9H ₂O 0.025g/L, FeC ₆H ₅O ₇0.003g/L, C ₆H ₈O ₇H ₂O 0.003g/L; 7 ^#Nutrient solution: MgSO ₄7H ₂O 0.125g/L, Na ₂HPO ₄0.075g/L, CaCO ₃0.1g/L, 1%FeC ₆H ₅O ₇5H ₂O 0.5ml/L, 1%C ₆H ₈O ₇H ₂O 0.5ml/L Na ₂MoO ₄2H ₂O0.5ml/L, A ₅1ml/L, B ₆1ml/L.2, nutrient solution nutritive salt is dissolved in the sterile distilled water in the pure level of algae kind preparatory phase operational analysis; Enlarged culturing and production are cultivated and can be used chemical pure, are dissolved in the tap water that purification filtering is handled to get final product.Nutritive salt adds in strict accordance with order during preparation, stops sedimentary generation, then the nutrient solution for preparing is sterilized.3, the algae kind prepares: A, original seed, the field acquisition filament of delivering vegetables, clean the back through sterilization and prepare homogenate; The purifying of B, filament or frustule is cultivated, and uses the pure method of multistage branch to carry out purifying, till assorted bacterium and assorted algae (can sediments microscope inspection observe).C, the expansion production of hybrid seeds will divide pure algae kind to be inoculated into aseptic culture fluid, aerated culture in Glass Containers, preparation algae kind.4, frustule is cultivated, when hair weeds cells is cultivated in the mass-producing of ventilating, suitable mode is to cultivate step by step, reason is that a cultured cells life cycle is about 12-14d, in more than 10 days, can not reach the ideal effect by small homogenate cultivation, need constantly change nutrient solution (from less to more) and container (ascending), increase the concentration of culturing cell gradually, obtain the algae liquid of high density; Culture condition is 24～26 ℃ of temperature, illumination 40～50 μ mol.m ^-2s ^-1, light dark period 12h: 12h, gas flow 5L/ minute aerated culture.5, the real-time monitoring of culturing process, set up monitoring condition and the monitoring standard is: temperature 24-26 ℃, illumination 40～50 μ mol.m ^-2s ^-1, light dark period 12h: 12h, pH8.0-8.5; Optimum parameter according to the frond growth is adjusted culture condition in good time.The equipment that needs has thermometer, luxmeter, spectrophotometer, pH meter, microscope, computer and conventional laboratory equipment and technical support.Regularly carry out scientific and reasonable monitoring, all parameter collection are set up database in computer, are used to analyze the growing state of frustule; 6, results reach OD in algae liquid concentration _750nmValue uses 200 purpose bolting silk nets to gather in 0.5-1.0; 7, drying, algae mud dry below 45 ℃, air-dry or the oven dry all can; In nutrient solution, stay certain density algae liquid to make the algae kind when 8, gathering in the crops, change the nutrient solution of certain volume, can carry out cultured continuously.

The present invention has the following advantages and effect: equipment is simple, and is easy to operate, cleanliness without any pollution, and security is good, and is nutritious; Compare with other inventions of delivering vegetables simultaneously, the resulting product of this technology is not the artificial Fa vegetable or the substitute of delivering vegetables, but truly has the goods of delivering vegetables of nutrition of delivering vegetables and taste.Use this technology to produce hair weeds cells and can absorb carbon dioxide in air and fixing nitrogen; in atmosphere, discharge oxygen; the effect that purifies air is arranged; do not produce hazardous and noxious substances; the nutrient solution salinity is low, and is with short production cycle, just can finish logarithmic growth about 14 days; the efficient height is widely used in the town and country mass-producing and cultivates.

Embodiment

Embodiment:

Cultivate the step of hair weeds cells:

1, nutrient solution preparation: the order that the storage solution for preparing is added in the entry is: SODIUMNITRATE 496mg/L, dipotassium hydrogen phosphate 39mg/L, magnesium sulfate heptahydrate 75mg/L, Calcium dichloride dihydrate 36mg/L, nine water water glass 58mg/L, micro-metals PIV 3ml/L, soil extract 3ml/L, the nutrient solution for preparing approximately uses after the cooling in 60-90 minute with 118-122 ℃ of high temperature dry sterilization.

2, the preparation of frustule homogenate: at first the wild filament of delivering vegetables is used 95% alcohol solution dipping 0.5-1.0min, use distilled water wash 3-4 time again, washing is 2-3 time in the nutrient solution of sterilization, and the nutrient solution that re-uses sterilization soaks 8-10h; Next is to add a spot of quartz sand with glass homogenizer, and with the broken homogenate of filament, homogenate is used for cell cultures;

3, algae kind axenic purification, homogenate is inoculated in the aseptic culture fluid, places 24～26 ℃ illumination box to leave standstill cultivation, behind the cultivation 40d, microscopically is chosen single filament or unicellular in sterilisable chamber, with aseptic culture fluid washing 4-5 time, leave standstill again and cultivate, repeat this operation 5-8 time, obtain the aseptic algae kind of this algae, with dividing pure algae kind to be inoculated into aseptic culture fluid, in Glass Containers, pass to filtrated air and cultivate, prepare the enlarged culturing algae kind of this algae;

4, frustule is cultivated

(1) cultivates place and incubator (the biological device photo-bioreactor of photo bio): select that the daylighting condition is good, water source cleaning, the water yield are abundant, the place of traffic convenience is as cultivating the place.(also can in plastic greenhouse, glasshouse or common house) installs bioreactor in the laboratory.Bioreactor has an injection port (to add nutrient solution and inoculum, top at reactor), leakage fluid dram (discharging nutrient solution, results algae mud is positioned at reactor bottom), ventage (bubbling air, keep cultivating and be in dynamically) at reactor bottom.

(2) the frustule liquid of algae kind original seed homogenate or axenic purification is inoculated in the bioreactor with the ratio of every liter of nutrient solution 1.0g weight in wet base, (stirs and increase CO from the ventage ventilation with 5L/ minute speed with the charge of air pump in culture vessel ₂Effect), air uses cotton and strainer to filter, and places 25 ℃ of temperature, illumination 40～50 μ mol.m ^-2s ^-1, cultivate under the condition of light dark period 12h: 12h

5, monitoring in real time;

Monitoring condition and the standard set up: temperature 24-26 ℃, illumination 40～50 μ mol.m ^-2s ^-1, light dark period 12h: 12h, pH8.0-8.5, other microorganisms such as bacterium, fungi and assorted bacterium detect: (1) is coated with flat board, nutrient solution is coated on the minimum medium of microbial culture, and whether more than 37 ℃ of incubation 24h, observing has bacterium colony to occur; Whether (2) microscopically is observed has assorted algae to exist.Standard Methods for Water and Waste Water one book that the nutrient concentration detection method is published referring to the U.S..

6, results; Reach OD in algae liquid concentration _750nmWhen being 0.6, the algae liquid of cultivating is discharged from the leakage fluid dram of bioreactor, use 200 purpose bolting silk nets to collect, collected algae mud is exactly our target product.

7, drying; Algae mud is air-dry at 25 ℃.

8, continue to cultivate: the culture vessel after collecting algae mud keeps a part of algae liquid, changes certain culture liquid and can be used for continuing to cultivate.

Claims (1)

1, a kind of method of cultivating hair weeds cells comprises the following steps:

A, nutrient solution preparation, the order that the storage solution for preparing is added in the entry is, SODIUMNITRATE 490-500mg/L, dipotassium hydrogen phosphate 35-40mg/L, magnesium sulfate heptahydrate 70-75mg/L, Calcium dichloride dihydrate 35-36mg/L, nine water water glass 55-60mg/L, micro-metals PIV3ml/L, soil extract 3ml/L, the nutrient solution for preparing uses after the cooling in 60-90 minute with 118-122 ℃ of high temperature dry sterilization;

The preparation of B, frustule homogenate, at first will deliver vegetables filament with 95% alcohol solution dipping 0.5-1.0min, use distilled water wash 3-4 time again, washing is 2-3 time in the nutrient solution of sterilization, the nutrient solution that re-uses sterilization soaked 8-10 hour, filament is placed in the glass homogenizer, adds quartz sand, grind to form slurry like material;

C, algae kind axenic purification, homogenate is inoculated in the aseptic culture fluid, places 24～26 ℃ illumination box to leave standstill cultivation, behind the cultivation 40d, microscopically is chosen single filament or unicellular in sterilisable chamber, with aseptic culture fluid washing 4-5 time, leave standstill again and cultivate, repeat this operation 5-8 time, obtain the aseptic algae kind of this algae, with dividing pure algae kind to be inoculated into aseptic culture fluid, in Glass Containers, pass to filtrated air and cultivate, prepare the enlarged culturing algae kind of this algae;

D, frustule are cultivated, and culture condition is 24～26 ℃ of temperature, illumination 40～50 μ mol.m ^-2s ^-1, light dark period 12h: 12h, aerated culture about gas flow 5L/ minute;

E, monitoring in real time, monitoring condition and standard are: temperature 24-26 ℃, illumination 40～50 μ mol.m ^-2s ^-1, light dark period 12h: 12h, pH8.0-8.5;

F, results reach OD in algae liquid concentration _750nmValue is gathered in 0.5-1.0, uses 200 purpose bolting silk nets to gather;

G, drying, algae mud dry below 45 ℃, air-dry or the oven dry;

H, continuation are cultivated, and the culture vessel after collecting algae mud keeps algae liquid, change nutrient solution, continue cultivation.