CN103805515B - A kind of station-service power source substratum and cultural method - Google Patents
A kind of station-service power source substratum and cultural method Download PDFInfo
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Abstract
The invention belongs to field of microalgae cultivation, disclose a kind of station-service power source improved culture medium formula and simple and easy culture technique.The cultivation of station-service power source uses f/2 substratum traditionally and uses open cement pool to cultivate, and the frustule doubling time is long, and the speed of growth is slow, and investment is large, yields poorly, easily pollutes, troublesome poeration.The substratum using me to invent also uses mineral water bottle cultivation station-service power source cell unsaturated fatty acid content high, cost is low, not easily pollute, easy handling, greatly reduce workload, and I adds the nutritive ingredients such as sodium bicarbonate, ironic citrate, potassium sorbate, glucose, ooze extract, soil Extract, complexing agent ethylenediamine tetraacetic acid (EDTA), cow dung and sheep excrement leach liquor in the medium, fertilizer and inorganic fertilizer used in combination, nutrition is more comprehensive, the station-service power source speed of growth increases substantially, culture cycle shortens, output increased 92%.
Description
Technical field
The invention belongs to field of microalgae cultivation, particularly relate to a kind of station-service power source substratum and cultural method.
Background technology
Station-service power source Dicrateriazhanjiangensis, is subordinate to Chrysophyta Chrysophyta, Chrysomonadales Chrysomonadales, Deng Bian algae section Isochrysidaceae, and Dicrateria inornata belongs to Dicrateria, sized by the unicellular algae that swims of 2-7 micron.Because of its acellular wall, be rich in unsaturated fatty acids and ecological suitability is strong, fast growth, is suitable for large-scale breeding, is considered to the desirable bait of marine animal artificial breeding, as the high-quality starter diet of the famous and precious fishery products such as mussel, oyster, mud blood clam, prawn.
The existence form of inorganic carbon in sea water is with HCO
3 -be main (90%), and free CO
2less than 1%.Past, people thought always, free CO in water body
2it is the unique forms that algae utilizes external source inorganic carbon.The work of Zhang Xuecheng shows, adds 50-100mmol/LNaHCO in Spirulina culture medium
3, not only can meet the needs of growth of spirulina platensis, and contribute to the pH value keeping frond growth.Woods Huimin is thought, NaHCO
3can be used as the carbon source of algal grown.My research shows that Dicrateria inornata can utilize HCO
3 -as carbon source.
Temperature, illumination, salinity, nutritive salt etc. have material impact to station-service power source growth.Nitrogen is the fundamental element forming amino acid, purine, pyrimidine, porphyrin, aminosugar and amine compound etc. in cellular metabolism, and thus nitrogen is that station-service power source grows most important nutritive element.My research display urea is used as nitrogenous source and no matter is all better than NaNO to the effect of the aspect such as increase, the synthesis of unsaturated fatty acids, the accumulation of astaxanthin of cell density
3and NH
4cl.In people's urine, 95% is water, and 5% is soluble inorganic salt, and wherein about 2% is urea.In the nitrogen metabolism of frustule, nitrogen must generate amino acid, resynthesis protein with the form of NH4+ and intracellular carbohydrate-derived ketone acid effect.Verified many algaes contain urease, and under its katalysis, urea decomposition goes out ammonia and utilized by micro-algae, and the difference of enzyme function causes various micro-algae to utilize the major cause of urea capacity variance.Sun Lingyi points out that the utilization of station-service power source to ammonium salt and urea is better than nitrate.It is best with urea effect that the research such as Zhang Guijie points out to cultivate station-service power source, and SODIUMNITRATE is taken second place, and ammonium chloride is the poorest.Zhang Qingtian etc. think with urea to be that nitrogenous source cultivation station-service power source effect is better than ammonium chloride.My research confirms that the urine of adding 1-2mL in often liter of nutrient solution has better promoter action to station-service power source growth.
The cultivation of station-service power source uses f/2 substratum traditionally and uses open cement pool to cultivate, and the frustule doubling time is long, and the speed of growth is slow, and investment is large, yields poorly, easily pollutes, troublesome poeration.The substratum using me to invent also uses mineral water bottle cultivation station-service power source cell unsaturated fatty acid content high, cost is low, not easily pollute, easy handling, greatly reduce workload, and I adds the nutritive ingredients such as sodium bicarbonate, ironic citrate, potassium sorbate, glucose, ooze extract, soil Extract, complexing agent ethylenediamine tetraacetic acid (EDTA), cow dung and sheep excrement leach liquor in the medium, fertilizer and inorganic fertilizer used in combination, nutrition is more comprehensive, the station-service power source speed of growth increases substantially, culture cycle shortens, output increased 92%.
Summary of the invention
In order to overcome the deficiency of current technology, the invention provides a kind of station-service power source substratum, its formula is as follows:
NH
4nO
325 milligrams, NaNO
35 milligrams, 5 milligrams, urea, KH
2pO
41.8 milligrams, Na
2siO
38 milligrams, NaHCO
3500 milligrams, 920 plant growth substance 8 international unit, potassium sorbate 10 milligrams, glucose 50 milligrams, propylene glycol alginate 15 milligrams, Sugaron 20 milligrams, propolis 9 milligrams, Sodium.alpha.-hydroxypropionate-L5 milligram, maltose alcohol 8 milligrams, stearoyl lactate 8 milligrams, ferric pyrophosphate 4 milligrams, Tripotassium Citrate 6 milligrams, sodium acetate 5 milligrams, D-sorbitol solution 1 milliliter, Tripolyglycerol monostearates 10 milligrams, people urinates 1 milliliter, ooze extracts liquid 1 milliliter, soil Extract 2 milliliters, cow dung leach liquor 1.5 milliliters, sheep excrement leach liquor 1.2 milliliters, f/2 trace element solution 1 milliliter, f/2 vitamin solution 1 milliliter, boil 1000 milliliters, cooled pure seawater,
Wherein f/2 trace element solution formula: ZnSO
44H
2o23 milligram, MnCl
24H
2o178 milligram, CuSO
45H
2o10 milligram, FeC
6h
5o
75H
2o3.9 gram, Na
2moO
42H
2o7.3 milligram, CoCl
26H
2o12 milligram, Na
2eDTA4.35 gram, distilled water 1000 milliliters,
F/2 vitamin solution is filled a prescription: vitamins B
120.5 milligram, vitamins B
1100 milligrams, vitamin H 0.5 milligram, distilled water 1000 milliliters.
A kind of station-service power source Simple culture method, concrete steps are as follows:
Culture vessel is home-use mineral water bottle, volume about 20 liters, water white transparency or band light sky blue, wash, rear distilled water rinse 3 times, the substratum 12 liters described in loading with funnel, fully shake up 5 minutes, select vigorous, the free of contamination algae liquid of vitality to inoculate, inoculum density 10 × 10
4-15 × 10
4cell/mL, bottleneck inserts two clean Glass tubings, and one is longer, inserts comparatively dark, at the bottom of bottle about 6 centimetres, in order to inflation; Another root is shorter, and about 15 centimetres, bottleneck is exposed at two ends, in order to exhaust; Two same thicknesses of Glass tubing, diameter 6 millimeters; Finally use cotton ball soaked in alcohol wiping bottleneck, seal with clean scotch tape and two Glass tubings are fixed; Bottleneck 7-10 centimetre is exposed in long Glass tubing upper end, is connected with air compressor or air pump with emulsion tube or plastic rubber material pipe, inflation, and aeration quantity should not be too large, can not float with frustule; Indoor cultivation, air-conditioning temperature control, optimum temps 27 ± 2 DEG C, fluorescent lamp irradiates, intensity of illumination 10000-12000lx, and light/dark cycle is 16L/8D, i.e. illumination 16 hours, dark 8 hours; Be cultured to the 6th day, stop inflation, open scotch tape, add 3 liters of fresh cultures with funnel from bottleneck, adhesive tape seals again, inflation, and continue to cultivate, to the 10th day, density was increased to 400 × 10
4-500 × 10
4cell/mL, can stop cultivate, pour out algae liquid from bottleneck, after being washed by mineral water bottle, add disinfection of Chinese drug liquid disinfectant 10-30 minute, after enter next round cultivate;
Described Traditional Chinese medicine disinfectant: be made up of the material of following weight ratio: the root of large-flowered skullcap 7 ~ 10 parts, 3 ~ 5 parts, okra powder, Herba Houttuyniae 3 ~ 5 parts, rheum officinale 5 ~ 10 parts, 5 ~ 10 parts, Radix Glycyrrhizae; Above-mentioned medicinal material is put into heatable container, adds the pure water of 3 ~ 5 times of volumes, continue after boiling to boil 30 ~ 50 minutes, rear filtration, precipitate 1 ~ 2 hour, after get clear liquor; Described okra powder to clean up and okra cauline leaf, flower and seed after drying is crushed to 250 orders through supper micron mill, then stirs evenly, sterilizing gained.
Preferably, to the mineral water bottle described in selecting, carry out disinfection after washing, use ultraviolet lamp disinfection, sterilization 10-20 minute.
Preferably, to the mineral water bottle described in selecting, carry out disinfection after washing, use high pressure nitrogen to spray sterilization, during sterilization 5-10 divides.
Preferably, to the mineral water bottle described in selecting, carry out disinfection after washing, use high-pressure carbon dioxide to spray sterilization, sterilization 5-6 minute.
Preferably, to the mineral water bottle described in selecting, point two sterilized phases after washing, first use high pressure nitrogen to spray sterilization, sterilization 2-3 minute.High-pressure carbon dioxide is used to spray sterilization, sterilization 2-3 minute.Algae culture is carried out again after sterilization.
Beneficial effect: the cultivation of station-service power source uses f/2 substratum traditionally and uses open cement pool to cultivate, and the speed of growth is slow, yields poorly, easily pollutes, troublesome poeration.The substratum using me to invent and use mineral water bottle to cultivate station-service power source unsaturated fatty acid content is high, cost is low, not easily pollute, easy handling, greatly reduce workload, and I adds the nutritive ingredients such as sodium bicarbonate, ironic citrate, potassium sorbate, glucose, ooze extract, soil Extract, complexing agent ethylenediamine tetraacetic acid (EDTA), cow dung and sheep excrement leach liquor in the medium, fertilizer and inorganic fertilizer used in combination, nutrition is more comprehensive, the station-service power source speed of growth increases substantially, culture cycle shortens, output increased 92%.
Accompanying drawing explanation
Fig. 1 station-service power source Simple culture method figure.
Wherein Reference numeral is: 1 mineral water bottle; 2 gas-filled valves; 3 vapor pipes.
Embodiment
1) station-service power source of inoculation carries out purifying (this area ordinary method, as long as can realize purifying, such as soxhlet extraction) in advance, and during to guarantee to inoculate, algae kind is in exponential phase of growth;
2) 920 plant growth substances are white crystalline powder, are not easily dissolved in water, first fully can dissolve (generally every gram dissolves more than 1 hour with 50 ml ethanols or 60 degree of white wine) with alcohol, then be watered dilution by desired concn;
3) because of ironic citrate (FeC
6h
5o
75H
2o) comparatively indissoluble solution, can add a small amount of tap water low-grade fever on stove and, to 80-90 DEG C, and constantly be stirred to whole thawing;
4) various nutritive salt will add by the order provided in formula, to avoid chemical reaction;
5) various nutritive salt particularly f/2 trace element solution and f/2 vitamin solution all need to be made into mother liquor in advance and be placed in 4 DEG C of refrigerator cold-storages and save backup, do not exceed 7 days storage period;
6) all nutritive salt will shake bucket 15 minutes after adding, and can inoculate after nutritive salt is mixed; Inoculum density 10 × 10
4-15 × 10
4cell/mL;
7) people urinates preparation method: get people and urinate 1000 milliliters, it is for subsequent use that flask boils rear cooling;
8) sheep excrement leach liquor preparation method: get 10 kilograms, fresh sheep excrement, adds 1 kilogram, wheat bran, Semen Maydis powder 0.5 kilogram, 1 liter, tap water, abundant stirring, loads plastics bag and ferments 10 days, after get 2 kilograms and put into basin, add 6 liters of distilled water, abundant stirring 15 minutes, leaves standstill, 500 order silk cover filterings after 7 days, get filtrate heated and boiled 30 minutes, to kill microorganism wherein, cooling, leave standstill 72 hours, get supernatant liquor, be sheep excrement leach liquor, load grinding port plug reagent bottle, sealing, is placed in 4 DEG C of refrigerators for subsequent use;
9) cow dung leach liquor preparation method: get cow dung 50 kilograms, add 3 kilograms, wheat bran, Semen Maydis powder 1 kilogram, 4 liters, tap water, abundant stirring, pile coniform, spontaneous fermentation 15 days, rear stand lamellar, be exposed to the sun under sunlight 3 days, after get 3 kilograms and put into bucket, add 10 liters of distilled water, fully stir evenly, leave standstill, 400 order silk cover filterings after 48 hours, get filtrate heating, boil 45 minutes, kill bacterium wherein, mould, virus etc., cooling, precipitate 48 hours, get supernatant liquor, obtain cow dung leach liquor, load grinding port plug reagent bottle, sealing, be placed in 4 DEG C of refrigerators for subsequent use.
10) ooze extracts liquid and preparation method thereof: get the more fertile ooze that what is not black again, add 2 points of fresh water, stir with 1 part of mud, leave standstill 1-2 minute, get upper strata mud in aluminum pot, the amount adding 1 gram of NaOH by every 1000 milliliters of mud adds NaOH, boils 20-30 minute.Constantly need stir when boiling, boil and leave standstill that within 24 hours, to get supernatant liquor for subsequent use afterwards;
11) soil Extract preparation method: fetch earth earth 1 kilogram, adding distil water 1000 milliliters, then add NaOH2-3 gram, boils 120 minutes, cooled and filtered, is placed in dark place and leaves standstill 48 hours, get supernatant liquor for subsequent use by clear liquid;
12) pharmaceutical chemicals purity used all needs chemical pure rank.
A kind of station-service power source substratum, its formula is as follows:
NH
4nO
325 milligrams, NaNO
35 milligrams, 5 milligrams, urea, KH
2pO
41.8 milligrams, Na
2siO
38 milligrams, NaHCO
3500 milligrams, 920 plant growth substance 8 international unit, potassium sorbate 10 milligrams, glucose 50 milligrams, propylene glycol alginate 15 milligrams, Sugaron 20 milligrams, propolis 9 milligrams, Sodium.alpha.-hydroxypropionate-L5 milligram, maltose alcohol 8 milligrams, stearoyl lactate 8 milligrams, ferric pyrophosphate 4 milligrams, Tripotassium Citrate 6 milligrams, sodium acetate 5 milligrams, D-sorbitol solution 1 milliliter, Tripolyglycerol monostearates 10 milligrams, people urinates 1 milliliter, ooze extracts liquid 1 milliliter, soil Extract 2 milliliters, cow dung leach liquor 1.5 milliliters, sheep excrement leach liquor 1.2 milliliters, f/2 trace element solution 1 milliliter, f/2 vitamin solution 1 milliliter, boil 1000 milliliters, cooled pure seawater,
Wherein f/2 trace element solution formula: ZnSO
44H
2o23 milligram, MnCl
24H
2o178 milligram, CuSO
45H
2o10 milligram, FeC
6h
5o
75H
2o3.9 gram, Na
2moO
42H
2o7.3 milligram, CoCl
26H
2o12 milligram, Na
2eDTA4.35 gram, distilled water 1000 milliliters,
F/2 vitamin solution is filled a prescription: vitamins B
120.5 milligram, vitamins B
1100 milligrams, vitamin H 0.5 milligram, distilled water 1000 milliliters.
A kind of station-service power source Simple culture method, concrete steps are as follows:
Culture vessel is home-use mineral water bottle, volume about 20 liters, water white transparency or band light sky blue, wash, rear distilled water rinse 3 times, the substratum 12 liters described in loading with funnel, fully shake up 5 minutes, select vigorous, the free of contamination algae liquid of vitality to inoculate, inoculum density 10 × 10
4-15 × 10
4cell/mL, bottleneck inserts two clean Glass tubings, and one is longer, inserts comparatively dark, at the bottom of bottle about 6 centimetres, in order to inflation; Another root is shorter, and about 15 centimetres, bottleneck is exposed at two ends, in order to exhaust; Two same thicknesses of Glass tubing, diameter 6 millimeters; Finally use cotton ball soaked in alcohol wiping bottleneck, seal with clean scotch tape and two Glass tubings are fixed; Bottleneck 7-10 centimetre is exposed in long Glass tubing upper end, is connected with air compressor or air pump with emulsion tube or plastic rubber material pipe, inflation, and aeration quantity should not be too large, can not float with frustule; Indoor cultivation, air-conditioning temperature control, optimum temps 27 ± 2 DEG C, fluorescent lamp irradiates, intensity of illumination 10000-12000lx, and light/dark cycle is 16L/8D, i.e. illumination 16 hours, dark 8 hours; Be cultured to the 6th day, stop inflation, open scotch tape, add 3 liters of fresh cultures with funnel from bottleneck, adhesive tape seals again, inflation, and continue to cultivate, to the 10th day, density was increased to 400 × 10
4-500 × 10
4cell/mL, can stop cultivate, pour out algae liquid from bottleneck, after being washed by mineral water bottle, add disinfection of Chinese drug liquid disinfectant 10-30 minute, after enter next round cultivate;
Described Traditional Chinese medicine disinfectant: be made up of the material of following weight ratio: the root of large-flowered skullcap 7 ~ 10 parts, 3 ~ 5 parts, okra powder, Herba Houttuyniae 3 ~ 5 parts, rheum officinale 5 ~ 10 parts, 5 ~ 10 parts, Radix Glycyrrhizae; Above-mentioned medicinal material is put into heatable container, adds the pure water of 3 ~ 5 times of volumes, continue after boiling to boil 30 ~ 50 minutes, rear filtration, precipitate 1 ~ 2 hour, after get clear liquor; Described okra powder to clean up and okra cauline leaf, flower and seed after drying is crushed to 250 orders through supper micron mill, then stirs evenly, sterilizing gained.
Preferably, to the mineral water bottle described in selecting, carry out disinfection after washing, use ultraviolet lamp disinfection, sterilization 10-20 minute.
Preferably, to the mineral water bottle described in selecting, carry out disinfection after washing, use high pressure nitrogen to spray sterilization, sterilization 5-10 minute.
Preferably, to the mineral water bottle described in selecting, carry out disinfection after washing, use high-pressure carbon dioxide to spray sterilization, sterilization 5-6 minute.
Preferably, to the mineral water bottle described in selecting, point two sterilized phases after washing, first use high pressure nitrogen to spray sterilization, sterilization 2-3 minute.High-pressure carbon dioxide is used to spray sterilization, sterilization 2-3 minute.Algae culture is carried out again after sterilization.
Last it is noted that above embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to previous embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in previous embodiment, or carries out equivalent replacement to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of embodiment of the present invention technical scheme.
Claims (3)
1. a station-service power source substratum, is characterized in that formula is as follows:
NH
4nO
325 milligrams, NaNO
35 milligrams, 5 milligrams, urea, KH
2pO
41.8 milligrams, Na
2siO
38 milligrams, NaHCO
3500 milligrams, 920 plant growth substance 8 international unit, potassium sorbate 10 milligrams, glucose 50 milligrams, propylene glycol alginate 15 milligrams, Sugaron 20 milligrams, propolis 9 milligrams, Sodium.alpha.-hydroxypropionate-L5 milligram, maltose alcohol 8 milligrams, stearoyl lactate 8 milligrams, ferric pyrophosphate 4 milligrams, Tripotassium Citrate 6 milligrams, sodium acetate 5 milligrams, D-sorbitol solution 1 milliliter, Tripolyglycerol monostearates 10 milligrams, people urinates 1 milliliter, ooze extracts liquid 1 milliliter, soil Extract 2 milliliters, cow dung leach liquor 1.5 milliliters, sheep excrement leach liquor 1.2 milliliters, f/2 trace element solution 1 milliliter, f/2 vitamin solution 1 milliliter, boil 1000 milliliters, cooled pure seawater,
Wherein f/2 trace element solution formula: ZnSO
44H
2o23 milligram, MnCl
24H
2o178 milligram, CuSO
45H
2o10 milligram, FeC
6h
5o
75H
2o3.9 gram, Na
2moO
42H
2o7.3 milligram, CoCl
26H
2o12 milligram, Na
2eDTA4.35 gram, distilled water 1000 milliliters,
F/2 vitamin solution is filled a prescription: vitamins B
120.5 milligram, vitamins B
1100 milligrams, vitamin H 0.5 milligram, distilled water 1000 milliliters.
2. a station-service power source cultural method, is characterized in that concrete steps are as follows:
Culture vessel is home-use mineral water bottle, volume 20 liters, water white transparency or band light sky blue, wash, after use distilled water rinse 3 times, load substratum according to claim 1 12 liters with funnel, fully shake up 5 minutes, vigorous, the free of contamination algae liquid of vitality is selected to inoculate, inoculum density 10 × 10
4-15 × 10
4cell/mL, bottleneck inserts two clean Glass tubings, and one is longer, inserts comparatively dark, at the bottom of bottle 6 centimetres, in order to inflation; Another root is shorter, and long 15 centimetres, bottleneck is exposed at two ends, in order to exhaust; Two same thicknesses of Glass tubing, diameter 6 millimeters; Finally use cotton ball soaked in alcohol wiping bottleneck, seal with clean scotch tape and two Glass tubings are fixed; Bottleneck 7-10 centimetre is exposed in long Glass tubing upper end, is connected with air compressor or air pump with emulsion tube or plastic rubber material pipe, inflation, and aeration quantity should not be too large, can not float with frustule; Indoor cultivation, air-conditioning temperature control, optimum temps 27 ± 2 DEG C, fluorescent lamp irradiates, intensity of illumination 10000-12000lx, and light/dark cycle is 16L/8D, i.e. illumination 16 hours, dark 8 hours; Be cultured to the 6th day, stop inflation, open scotch tape, add 3 liters of fresh cultures with funnel from bottleneck, adhesive tape seals again, inflation, and continue to cultivate, to the 10th day, density was increased to 400 × 10
4-500 × 10
4cell/mL, namely stops cultivating, pours out algae liquid, washed by mineral water bottle from bottleneck, repeat aforesaid operations, namely enters next round and cultivates.
3. a station-service power source cultural method, it is characterized in that concrete steps are as follows: 1) culture vessel is home-use mineral water bottle, volume 20 liters, water white transparency or band light sky blue, wash, rear distilled water rinse 3 times, substratum according to claim 1 12 liters is loaded with funnel, fully shake up 5 minutes, select vigorous, the free of contamination algae liquid of vitality to inoculate, inoculum density 10 × 10
4-15 × 10
4cell/mL, bottleneck inserts two clean Glass tubings, and one is longer, inserts comparatively dark, at the bottom of bottle 6 centimetres, in order to inflation; Another root is shorter, and long 15 centimetres, bottleneck is exposed at two ends, in order to exhaust; Two same thicknesses of Glass tubing, diameter 6 millimeters; Finally use cotton ball soaked in alcohol wiping bottleneck, seal with clean scotch tape and two Glass tubings are fixed; Bottleneck 7-10 centimetre is exposed in long Glass tubing upper end, is connected with air compressor or air pump with emulsion tube or plastic rubber material pipe, inflation, and aeration quantity should not be too large, can not float with frustule; Indoor cultivation, air-conditioning temperature control, optimum temps 27 ± 2 DEG C, fluorescent lamp irradiates, intensity of illumination 10000-12000lx, and light/dark cycle is 16L/8D, i.e. illumination 16 hours, dark 8 hours; Be cultured to the 6th day, stop inflation, open scotch tape, add 3 liters of fresh cultures with funnel from bottleneck, adhesive tape seals again, inflation, and continue to cultivate, to the 10th day, density was increased to 400 × 10
4-500 × 10
4cell/mL, namely stop cultivate, pour out algae liquid from bottleneck, after being washed by mineral water bottle, add disinfection of Chinese drug liquid disinfectant 10-30 minute, after enter next round cultivate; Described Traditional Chinese medicine disinfectant: be made up of the material of following weight ratio: the root of large-flowered skullcap 7 ~ 10 parts, 3 ~ 5 parts, okra powder, Herba Houttuyniae 3 ~ 5 parts, rheum officinale 5 ~ 10 parts, 5 ~ 10 parts, Radix Glycyrrhizae; Above-mentioned medicinal material is put into heatable container, adds the pure water of 3 ~ 5 times of volumes, continue after boiling to boil 30 ~ 50 minutes, rear filtration, precipitate 1 ~ 2 hour, after get clear liquor; Described okra powder to clean up and okra cauline leaf, flower and seed after drying is crushed to 250 orders through supper micron mill, then stirs evenly, sterilizing gained.
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