CN1174090C - A kind of method of cultivating hair vegetable cell - Google Patents

Abstract

The invention discloses a method for cultivating hair vegetable cells. 1. Preparation of culture solution: the order of adding the prepared storage solution into water is sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate , sodium silicate nonahydrate, trace metal element solution PIV, and soil leaching solution are prepared in proportion; 2. The preparation of algae cell homogenate, first wash the filaments of wild hairy vegetables with ethanol and distilled water, soak in sterile culture solution for 8-10 Hours; followed by breaking the filament homogenate with a glass homogenizer, and using the homogenate for cell culture; 3. Aseptic purification of algal species; 4. Algal cell culture; 5. Real-time monitoring; 6. Harvesting; 7. Drying ; 8. Continue to cultivate. The method of the invention is simple, convenient to operate, safe and pollution-free, has a short growth period, high production efficiency, comprehensive nutritional components and high nutritional content, and is widely applicable to large-scale production and cultivation in urban and rural areas.

Description

A kind of method of cultivating hair vegetable cell

Technical field

The invention relates to the field of biotechnology, and more specifically relates to a method for cultivating Nostocchis cells.

Background technique

Nostoc is a kind of algae group with wide distribution, complex ecological environment and various morphological structures in the cyanophyta Cyanophyta Cyanophyceae Cyanophyceae segmental algae order Hormogonales Nostocaceae. , leaf-like, filamentous, bubble-like and other shapes, hollow or solid, floating or growing. Nostoc is the main component species in many extreme environments, traces of Nostoc can be found in extreme environments such as high temperature, severe cold, strong ultraviolet radiation, extremely low light intensity, and high salinity, some of which are such as Nostoc flagelliforme), Nostoc commune and Nostoc sphaeroides are nutritious and edible, and have certain medicinal value. Since ancient times, Facai, Ge Xianmi, and ground fungus have been the natural traditional foods of the Chinese nation. Among them, Facai has long been favored by consumers because of its rich nutrition and auspicious pronunciation (homophonic with "facai"), and it is listed in many ancient books. There are detailed records. Nostoc, also known as hair dish and ground hair, scientific name Nostoc hairy, is an edible terrestrial cyanobacteria distributed worldwide. It can often be seen in the desert and semi-desert areas of western and northwestern my country. Its edible and medicinal history can be Dating back to 3,000 years ago, there have been some studies on its morphology, ecology, physiology and developmental biology in recent decades. Nostocchi has functions such as diet therapy, health care, and nourishment, and has been sold at home and abroad. The contradiction between environmental degradation and grassland protection brought about by the current resource development has become increasingly acute. Some studies have said that mining 1 kg of Nostoc can destroy 3 hectares For the grassland, it can be seen that the damage to the ecological environment and the shrinkage and decline of natural resources caused by excessive mining are extremely serious. There is an irreconcilable contradiction between the huge market demand and the limited amount of resources, and the only way to solve it is to carry out artificial cultivation and intensive breeding of Nostocs. Because the growth of Nostocs is distributed in the desert, semi-desert and grasslands, the living environment has certain specificity, and the conditions required for its growth, such as temperature, moisture, light, light-dark cycle, nutrition, etc., are strict, and it is currently difficult to artificially cultivate mature There is no report of successful continuous culture of Nostocchia filaments, even Nostocys cells.

Contents of the invention

The object of the present invention is to provide a kind of method for cultivating Nostocchilloides cells, in order to obtain artificial cultures with basic Nostocysts nutrient components under the situation that filamentous body is difficult to artificially cultivate, the method is simple and easy to operate, and the cultured With the nutritional content of Nostocs, various health drinks can be made to alleviate the market demand for Nosts.

In order to achieve the above object, the present invention has taken the following technical measures: 1, the cultivation first considers the formula of the nutrient solution, and the present invention compares and screens out the nutrient solution formula most suitable for the cultivation of Nostocella cyanobacteria from 8 nutrient solutions suitable for the growth of cyanobacteria It is HGZ culture solution, NaNO ₃ 490-500mg/L, K ₂ HPO ₄ 35-40mg/L, MgSO ₄ 7H ₂ O 70-75mg/L, CaCl ₂ 2H ₂ O 35-36mg/L, Na ₂ SiO ₄ 9H ₂ O 55-60mg/L, PIV 3ml/L, soil extract 3ml/L. The following are the formulas of the other 7 culture solutions: 1 ^#Culture solution: Ca(NO ₃ ) ₂ 0.2g/L, K ₂ HPO ₄ 0.075g/L, MgSO ₄ 7H ₂ O 0.1g/L, Na ₂ CO ₃ 0.2g/L, 0.5% Na ₂ MoO ₄ 4ml, 0.5% HBO ₃ 4ml, 1% FeC ₆ H ₅ O ₇ 5H ₂ O 0.5ml, 1% C ₆ H ₈ O ₇ H _{2 O} 0.5ml/L ; 2 ^# Culture solution: CuSO ₄ ·5H ₅ O 19.6mg/L, ZnSO ₄ ·7H ₂ O 44.0mg/L, CoCl ₂ ·6H ₂ O 20.0mg/L, MnCl ₂ ·4H ₂ O 36.0mg/L, Na ₂ MoO ₄ 2H ₂ O 12.6mg/L, H ₃ BO ₃ 618.4mg/L; 3 ^# culture solution: 1 ^# : 2 ^# = 1: 1000; 4 ^# culture solution: MgSO ₄ 7H ₂ O 0.2g /L, K ₂ HPO ₄ 0.5g/L, NaCl 0.2g/L, CaSO ₄ 2H ₂ O 0.1g/L, FeCl ₃ 6H ₂ O 0.005g/L, CaCO ₃ 1.0g/L; 5# culture Solution: MgSO ₄ 7H ₂ O 125mg/L, KNO ₃ 250mg/L, CaCl ₂ 27mg/L, NaOH 3.76mg/L, FeSO ₄ 7H ₂ O 1.0mg/L, A ₅ 1.0ml; 6 ^# culture solution : Ca(NO ₃ ) ₄ 4H ₂ O 0.04g/L, K ₂ HPO ₄ 0.01g/L, MgSO ₄ 7H ₂ O 0.025g/L, Na ₂ CO ₃ 0.02g/L, Na ₂ SiO ₃ 9H ₂ O 0.025g/L, FeC ₆ H ₅ O ₇ 0.003g/L, C ₆ H ₈ O ₇ H ₂ O 0.003g/L; 7 ^# culture solution: MgSO ₄ 7H ₂ O 0.125g/L, Na ₂ HPO ₄ 0.075g/L, CaCO ₃ 0.1g/L, 1% FeC ₆ H ₅ O ₇ 5H ₂ O 0.5ml/L, 1% C ₆ H ₈ O ₇ H ₂ O 0.5ml/L Na _{2MoO 4} ·2H ₂ O 0.5ml/L, A ₅ 1ml/L, B ₆ 1ml/L. 2. Nutrient salts in the culture medium should be of analytical grade in the algae preparation stage and dissolved in sterile distilled water; chemically pure can be used for expanded cultivation and production cultivation and dissolved in tap water that has been purified and filtered. Nutrient salts are added in strict order during preparation to prevent precipitation, and then the prepared culture solution is sterilized. 3. Preparation of algae species: A, original species, collect Nostocella filaments in the field, prepare homogenate after disinfection and cleaning; B, purify and cultivate algae filaments or algae cells, use multi-fraction pure method to purify until Until there are no bacteria and algae (can be observed under a microscope). C. Expand seed production, inoculate the pure algae seeds into the sterile culture solution, and culture them in a glass container with ventilation to prepare the algae seeds. 4. For algae cell culture, when carrying out aeration and large-scale cultivation of Nostocella cells, the appropriate way is to cultivate them step by step, because the life cycle of cells cultured at one time is about 12-14 days, and the cultured by tiny homogenate will not change within more than 10 days. To achieve the desired effect, it is necessary to continuously replace the culture medium (from small to large) and containers (from small to large), and gradually increase the concentration of cultured cells to obtain high-concentration algae liquid; the culture conditions are temperature 24-26°C, Light 40～50μmol.m ^-2 s ^-1 , light-dark cycle 12 hours: 12 hours, gas flow 5L/min aeration culture. 5. Real-time monitoring of the cultivation process, the establishment of monitoring conditions and monitoring standards are: temperature 24-26 ° C, light 40-50 μmol.m ^-2 s ^-1 , light-dark cycle 12 hours: 12 hours, pH 8.0-8.5; The optimal parameters for the growth of algae were to adjust the culture conditions in due course. The equipment needed is thermometer, illuminance meter, spectrophotometer, pH meter, microscope, computer as well as general laboratory equipment and technical support. Scientific and reasonable monitoring is carried out regularly, and all parameters are collected into the computer to establish a database for analyzing the growth of algae cells; 6. Harvesting, when the concentration of algae liquid reaches OD _750nm value of 0.5-1.0, use 200-mesh silk screen Harvesting; 7. Drying, the algae mud can be dried below 45°C, air-dried or dried; 8. When harvesting, leave a certain concentration of algae liquid in the culture medium as algae, and replace a certain volume of culture liquid, which can for continuous cultivation.

The present invention has the following advantages and effects: simple equipment, convenient operation, clean and pollution-free, good safety, and rich nutrition; at the same time, compared with other inventions of hair vegetables, the products obtained by this technology are not artificial hair vegetables or substitutes for hair vegetables. It is a Nostoc food product that has the nutrition and taste of Nostoc in the true sense. Using this technology to produce hair vegetable cells can absorb carbon dioxide in the air and fix nitrogen, release oxygen into the atmosphere, have the effect of purifying the air, do not produce toxic and harmful substances, the culture medium has low salinity, and the production cycle is short, about 14 days. Complete logarithmic growth, high efficiency, widely applicable to large-scale cultivation in urban and rural areas.

Detailed ways

Example:

Steps for culturing Nostocia cells:

1. Culture solution preparation: The order of adding the prepared storage solution to water is: sodium nitrate 496mg/L, dipotassium hydrogen phosphate 39mg/L, magnesium sulfate heptahydrate 75mg/L, calcium chloride dihydrate 36mg/L, nine Sodium silicate water 58mg/L, trace metal elements PIV 3ml/L, soil extract 3ml/L, the prepared culture solution is sterilized by high temperature dry heat at 118-122°C, cooled for about 60-90 minutes before use.

2. Preparation of algae cell homogenate: first soak the wild hairy vegetable filaments in 95% ethanol solution for 0.5-1.0 minutes, then wash 3-4 times with distilled water, and wash 2-3 times in sterilized culture solution. Then use sterilized culture solution to soak for 8-10 hours; followed by adding a small amount of quartz sand with a glass homogenizer to break and homogenize the filaments, and the homogenate is used for cell culture;

3. Aseptically purify the algal species, inoculate the homogenate into the sterile culture medium, and place it in a light incubator at 24-26°C for static cultivation. After culturing for 40 days, pick out a single algal filament or algae under a microscope in a sterile room. For single cells, wash 4-5 times with aseptic culture medium, carry out static recultivation, repeat this operation 5-8 times, obtain the sterile algae species of the algae, inoculate the pure algae species into the aseptic culture medium, Cultivate the algae in a glass container by filtering the air to prepare the algae species for expanding cultivation;

4. Algae cell culture

(1) Cultivation site and incubator (photo-bioreactor): Choose a place with good lighting conditions, clean water source, abundant water, and convenient transportation as the training site. Install photobioreactors in laboratories (also in plastic greenhouses, glass greenhouses or ordinary houses). The photobioreactor has a sample inlet (adding culture solution and inoculum, in the upper part of the reactor), a liquid outlet (discharging the culture solution, harvesting algae sludge, located at the bottom of the reactor), and a vent (introducing air , keep the culture in dynamic, at the bottom of the reactor).

(2) Inoculate the homogenate of the original algae seed or the sterile purified algae cell liquid in the photobioreactor at a ratio of 1.0 g wet weight per liter of culture liquid, and use an air pump in the culture container at a speed of 5 L/min Air is ventilated from the vent (to agitate and increase CO ₂ ), the air is filtered with cotton and a filter, and placed at a temperature of 25°C, with a light of 40-50 μmol.m ^-2 s ^-1 , and a light-dark cycle of 12 hours: 12 hours cultivated under the conditions of

5. Real-time monitoring;

Established monitoring conditions and standards: temperature 24-26°C, light 40-50 μmol.m ^-2 s ^-1 , light-dark cycle 12 hours: 12 hours, pH 8.0-8.5, detection of other microorganisms such as bacteria, fungi and miscellaneous bacteria (1) Smear a flat plate, spread the culture solution on the basic medium for bacterial culture, incubate at 37°C for more than 24 hours, and observe whether there are colonies; (2) Observe under a microscope whether there are miscellaneous algae. For the detection method of nutrient salt concentration, refer to the book Standard Methods for Water and Waste Water published in the United States.

6. Harvesting: when the concentration of the algae liquid reaches OD _750nm of 0.6, the cultured algae liquid is discharged from the discharge port of the photobioreactor and collected with a 200-mesh silk screen. The collected algae mud is our target product .

7. Drying; the algae mud is air-dried at 25°C.

8. Continue to cultivate: Keep a part of the algae liquid in the culture container after collecting the algae mud, and replace a certain amount of culture liquid to continue the culture.

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 0.5-1.0 minute, 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 12 hours: 12 hours, 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 12 hours: 12 hours, 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.