CN107646693B - Production method of golden yellow nostoc - Google Patents
Production method of golden yellow nostoc Download PDFInfo
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- CN107646693B CN107646693B CN201711113508.2A CN201711113508A CN107646693B CN 107646693 B CN107646693 B CN 107646693B CN 201711113508 A CN201711113508 A CN 201711113508A CN 107646693 B CN107646693 B CN 107646693B
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- 241000192656 Nostoc Species 0.000 title claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 title claims abstract description 14
- 241000452732 Nostoc sphaeroides Species 0.000 claims abstract description 76
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- 230000006698 induction Effects 0.000 claims abstract description 22
- 239000002609 medium Substances 0.000 claims abstract description 14
- 230000001954 sterilising effect Effects 0.000 claims abstract description 14
- 239000002054 inoculum Substances 0.000 claims abstract description 10
- 239000011159 matrix material Substances 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 238000005286 illumination Methods 0.000 claims abstract description 6
- 238000005273 aeration Methods 0.000 claims abstract description 5
- 230000003321 amplification Effects 0.000 claims abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 229910021577 Iron(II) chloride Inorganic materials 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 9
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 9
- 239000011565 manganese chloride Substances 0.000 claims description 9
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 8
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 8
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 239000011684 sodium molybdate Substances 0.000 claims description 6
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 239000011686 zinc sulphate Substances 0.000 claims description 6
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- 238000001914 filtration Methods 0.000 claims description 4
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 230000008635 plant growth Effects 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 239000008213 purified water Substances 0.000 claims description 2
- 239000002352 surface water Substances 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 claims description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims 2
- 229910000831 Steel Inorganic materials 0.000 claims 1
- 239000002243 precursor Substances 0.000 claims 1
- 239000010959 steel Substances 0.000 claims 1
- 235000021466 carotenoid Nutrition 0.000 abstract description 5
- 150000001747 carotenoids Chemical class 0.000 abstract description 5
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- 238000005406 washing Methods 0.000 description 6
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
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- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 description 2
- 229930002875 chlorophyll Natural products 0.000 description 2
- 235000019804 chlorophyll Nutrition 0.000 description 2
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 2
- 230000004438 eyesight Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
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- 108060006184 phycobiliprotein Proteins 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
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- 241000894006 Bacteria Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 208000001140 Night Blindness Diseases 0.000 description 1
- 241000692932 Nostoc flagelliforme Species 0.000 description 1
- 235000021529 Nostoc flagelliforme Nutrition 0.000 description 1
- 241000606333 Phos Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 206010038084 Rectocele Diseases 0.000 description 1
- CGZKSPLDUIRCIO-UHFFFAOYSA-N Scytonemin Natural products OC1=CC=C(C=C1)C=C1C(C(=C2C1=NC=1C=CC=CC2=1)C=1C(C(C2=NC=3C=CC=CC=3C2=1)=CC1=CC=C(C=C1)O)=O)=O CGZKSPLDUIRCIO-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 206010053615 Thermal burn Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
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- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
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- 230000002068 genetic effect Effects 0.000 description 1
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- 229910052732 germanium Inorganic materials 0.000 description 1
- GNPVGFCGXDBREM-UHFFFAOYSA-N germanium atom Chemical compound [Ge] GNPVGFCGXDBREM-UHFFFAOYSA-N 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- 101150018863 maa gene Proteins 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
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- 229920001282 polysaccharide Polymers 0.000 description 1
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- 230000005855 radiation Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- CGZKSPLDUIRCIO-RPCRKUJJSA-N scytonemin Chemical compound C1=CC(O)=CC=C1\C=C\1C2=NC3=CC=CC=C3C2=C(C=2C(C(=C/C=3C=CC(O)=CC=3)/C=3C=2C2=CC=CC=C2N=3)=O)C/1=O CGZKSPLDUIRCIO-RPCRKUJJSA-N 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 239000010454 slate Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229910052712 strontium Inorganic materials 0.000 description 1
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
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Images
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of algae culture, and particularly relates to nostoc sphaeroides culture, in particular to a production method of nostoc sphaeroides golden yellow. The production method comprises the following steps: 1) seed production: selecting nostoc sphaeroids kutz matrix, sterilizing to prepare an inoculum, and transferring the inoculum into a sterilized first culture medium for aeration culture; 2) amplification culture: when the diameter of the nostoc sphaeroides is 0.5-1.0 mm, transferring the nostoc sphaeroides into a culture container with a first induction medium for open culture according to the ratio of the nostoc sphaeroides culture to the first induction medium in the step 1) to be 1: 5-50; 3) induction: transferring the nostoc sphaeroides with the diameter of 1.5-2.5 mm to a second induction culture medium, and culturing for 30-60 days by illumination to obtain the nostoc sphaeroides with the diameter of 4-6 mm. The invention improves the proportion of carotenoid in nostoc sphaeroides cells by regulating and controlling nutrient salt, light intensity and light quality, so that the nostoc sphaeroides cells are golden yellow, the variety of nostoc sphaeroides products is enriched, and the market demand is met.
Description
Technical Field
The invention belongs to the field of algae culture, and particularly relates to nostoc sphaeroides culture, in particular to a production method of nostoc sphaeroides golden yellow.
Background
Nostoc sphaeroides (Nostoc sphaeroides), a scientific name, is a fresh water microalgae belonging to the genus Nostoc of the family Nostoc of the class Cyanophyceae. It belongs to the same genus as Nostoc flagelliforme, but has a different form. The glue colony is spherical, has a diameter of several centimeters, grows among gravels of paddy fields, shallow ponds, lakes and streams in mountain areas, and is one of the earliest prokaryotes photosynthetic organisms on the earth.
Nostoc sphaeroids kutz is a famous local special product in Hubei, the protein content of Nostoc sphaeroids kutz reaches 56%, and phycobiliprotein in Nostoc sphaeroids kutz has strong antioxidant and anticancer effects. Nostoc sphaeroids kutz contains a large amount of blue algae polysaccharide with biological activity, and has the effects of inhibiting the growth of cancer cells, enhancing the immunity of organisms, resisting viruses and the like. The nostoc sphaeroides also contains 17 amino acids including 8 amino acids necessary for human body; in addition, VA and VB1、VB2VC, VE and other vitamins are also high in content, and the vitamin composition contains calcium, iron, iodine, strontium, phosphorus, sulfur, potassium, magnesium, barium, germanium, trace zinc, copper, manganese and other minerals.
Historically, there are many written records of the food of nostoc sphaeroides, and the detailed records of how to cook nostoc sphaeroides are recorded in the ' food bill with garden ' (1792), the ' vegetarian food saying a little ' of xu Baochen and the ' tuning ancient collection ' of the child ' recommended by the Qing dynasty. Nostoc sphaeroides appears in famous medical records of the pottery and Hongjing of the Liang Dynasty, and is then collected in Ben Cao gang mu Shi Yi (1765) of the Qing Dynasty Zhao academic Ming, Shen Xian Ji Shi Liang (1797) of the Bergenipin, Yao Bai Yao (1795) and nationwide Chinese herbal medicine compilation. The medicinal efficacy of nostoc sphaeroides can be summarized as follows: has cold property and light taste, and has effects of clearing heat, astringing, improving eyesight, invigorating qi, benefiting intestine and stomach, and can be used for treating nyctalopia, rectocele, burn, scald, etc., and can be taken for a long time to relieve fatigue, beautify skin, and prolong life.
Nostoc sphaeroides contains chlorophyll, carotenoid, phycobiliprotein, ultraviolet absorbing pigments (such as MAAs and Scytonemin), and the like, and the group of Nostoc sphaeroides presents a color as a result of the interaction of the pigments. Under the conventional culture conditions, the nostoc sphaeroides has high content of chlorophyll and phycoerythrin, so that the nostoc sphaeroides mostly presents dark green or black, which are two main colors of the commercially available nostoc sphaeroides. In the artificial culture process, the nostoc sphaeroides presents emerald green, red, blue, golden yellow and other colors at certain probability, but when the nostoc sphaeroides with special colors are used as parents to be continuously cultured, the progeny nostoc sphaeroides are restored to dark green or black, and the color property of the progeny nostoc sphaeroides cannot be maintained by the culture method known in the prior art. Since golden yellow has a cultural connotation of grandeur, graceful and luxurious, there is a strong demand for golden yellow nostoc in the market, and thus there is a need to provide a method capable of stably producing golden yellow nostoc.
Disclosure of Invention
Based on a large number of experiments, the inventors creatively found that the color trait of nostoc sphaeroides is not genetically mutable but induced, so that the progeny product with the desired color can be obtained by controlling the culture conditions of nostoc sphaeroides.
On the basis of the above findings, the present invention provides a production method of nostoc sphaeroides which is simple and easy to implement and can stably obtain nostoc sphaeroides with golden yellow.
Specifically, the production method of nostoc sphaeroides comprises the following steps:
1) seed production: selecting nostoc sphaeroids kutz matrix, sterilizing to prepare an inoculum, and transferring the inoculum into a sterilized first culture medium for aeration culture;
2) amplification culture: when the diameter of the nostoc sphaeroides is 0.5-1.0 mm, transferring the nostoc sphaeroides into a culture container with a first induction culture medium according to the ratio of the nostoc sphaeroides culture in the step 1) to carry out open culture, wherein the culture container is selected from a columnar photobioreactor, a plate photobioreactor, a box photobioreactor or a raceway pond type culture pond, and the first induction culture medium is 1: 5-50;
3) induction: transferring the nostoc sphaeroides with the diameter of 1.5-2.5 mm to a second induction culture medium, and culturing for 30-60 days by illumination to obtain the nostoc sphaeroides with the diameter of 4-6 mm.
By the method, the ratio of carotenoid in nostoc sphaeroides cells can be increased, and the nostoc sphaeroides can be golden yellow in appearance. In the step 2), the "nostoc sphaeroides culture of the step 1) means a culture product containing nostoc sphaeroides and the first medium.
The nostoc sphaeroids kutz matrix has the characteristics of good elasticity and strong toughness. Specifically, in the step 1), the nostoc sphaeroides as a matrix meets the following characteristics:
i) the shape is a regular sphere with the diameter of 3-10 mm, the dry matter content is 1-5 wt.%, and the color is dark green;
ii) falls from the height of 50cm through a free fall type, does not crack after being impacted with a hard horizontal plane, and rebounds to the height of 3-20 cm;
iii) the ability to pull the filaments out when broken.
Since the color characteristics of nostoc sphaeroides are not genetic variation but induced variation, the slight difference between the source of nostoc sphaeroides and the genetic information thereof does not influence the induced variation. The nostoc sphaeroides as the parent of the present invention is not particularly limited in its origin, and any nostoc sphaeroides can be used as long as it satisfies the above characteristics, regardless of the place of collection, the collection method, and the like. The "hard surface" is not particularly limited in hardness, and any surface of a general metal plate, slate, cement, or the like may be used as long as it is substantially rigid and has hardness higher than that of nostoc.
Further, in step 1), the sterilization mode for nostoc sphaeroides is selected from ultraviolet radiation sterilization, 75% (volume ratio) alcohol soaking sterilization or 0.1-1% (weight ratio) sodium hypochlorite solution soaking sterilization.
Further, in step 1), the conditions of the aeration culture are as follows:
i) uses air as raw material to increase its CO2The content is 0.1-5% (volume ratio), and the air is used as ventilation gas after filtration and sterilization;
ii) the culture temperature is 20-30 ℃;
iii) the light supply period is 24h, and the light intensity is 20-50 mu mol phos/(m)2S) using a light source selected from one or more of fluorescent lamps, LED lamps, plant growth lamps or sunlight.
Further, in step 1), the first culture medium is composed of the following kinds and amounts of raw materials:
the water is selected from one or more of purified water, single distilled water, tap water, natural surface water and underground water.
Further, in step 1), the first culture medium is sterilized by filtration through a 0.2um pore size filter or by autoclaving.
Further, in the step 1), the nostoc sphaeroides parent is obtained by screening wild species or is filial generation within five generations obtained by the production method.
Further, in step 2), the first induction medium is based on the first medium, and NaNO is omitted3、Ca(H2PO4)2、MgK2(SO4)2、NaHCO3Or FeCl2One or more of the components (a).
Further, in step 2), the second induction medium is based on the first induction medium, and NaNO is omitted3、Ca(H2PO4)2、MgK2(SO4)2、NaHCO3、FeCl2、H3BO3、MnCl2·4H2O、ZnSO4·7H2O or Na2MoO4·2H2One or more components of O.
Further, in the step 3), the light intensity of the illumination culture is more than 100 mu mol photosns/(m)2S), preferably 100-2S) or controlling the wavelength of light to be 400-550 nm. The method can be realized by changing a light source, adding a special filter film and the like.
The invention has the beneficial effects that:
1. the inventor creatively discovers that the color character of nostoc sphaeroides is not heredity mutation but inductivity mutation, and provides a method for realizing stable mass production of nostoc sphaeroides.
2. The invention improves the proportion of carotenoid in nostoc sphaeroides cells by regulating and controlling nutrient salt, light intensity and light quality, so that the nostoc sphaeroides cells are golden yellow, the variety of nostoc sphaeroides products is enriched, and the market demand is met.
3. The nostoc sphaeroides obtained by the production method has high carotenoid content, so that the effects of resisting oxidation, regulating immunity, delaying senescence and protecting eyesight are greatly enhanced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
FIG. 1 is a photograph of a wild species of Nostoc sphaeroides.
FIG. 2 is a photograph of the nostoc sphaeroides which is prepared by the production method provided by the invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The nostoc sphaeroides used in this example was collected from Hubei Hefeng Famazhen rice field and purified in this laboratory to obtain purified algal species. Selecting dark green globular nostoc sphaeroides (phi 4mm) with good elasticity and strong toughness as a matrix, sequentially washing with tap water for 20min, sufficiently washing with sterile water for 10min, irradiating with UVB ultraviolet lamp for 10min, soaking in 0.15% sodium hypochlorite solution for 2min, washing with sterile water for 3 times, and making into inoculum.
The first medium was prepared according to the following recipe: NaNO30.21g/L，Ca(H2PO4)20.12g/L，MgK2(SO4)20.24g/L，NaHCO30.15g/L,FeCl210mg/L，Na2EDTA2.4mg/L，H3BO33mg/L，MnCl2·4H2O1.50mg/L，ZnSO4·7H2O 0.16mg/L，CuSO4·5H2O 0.08mg/L，Na2MoO4·2H2O 0.4mg/L，CoCl2·6H2O0.04 mg/L. The culture medium is prepared by sterilizing with distilled water at 121 deg.C under 103.5kPa for 20min, and cooling.
The inoculum was inoculated into the sterilized first medium and subjected to aerobic culture under the conditions of 40. mu. mol photons/(m 2. s) daylight lamp, 23 ℃ and 24h illumination.
After 30 days, obtaining a population with the diameter of 1mm, and transferring the population into a 40L cylindrical photobioreactor for culture according to the ratio of 1: 10; the culture medium used is a first induction culture medium, and comprises the following components: FeCl210mg/L，Na2EDTA2.4mg/L，H3BO33mg/L，MnCl2·4H2O 1.50mg/L，ZnSO4·7H2O 0.16mg/L，CuSO4·5H2O 0.08mg/L，Na2MoO4·2H2O0.4mg/L，CoCl2·6H2O0.04 mg/L. Open culture was carried out at 24h at 23 ℃ in 50. mu. molphostons/(m 2. multidot.s).
After 15 days, the diameter of the nostoc sphaeroides reaches 2.0mm, and the nostoc sphaeroides is transferred to a 500L cylindrical photobioreactor for culture, wherein the culture medium is a second induction culture mediumThe composition is as follows: FeCl210mg/L，Na2EDTA2.4mg/L，H3BO33mg/L，MnCl2·4H2O 1.50mg/L，CuSO4·5H2O 0.08mg/L，CoCl2·6H2O0.04 mg/L. Adopting a blue-green LED lamp as a light source, and culturing for 45 days to obtain the nostoc sphaeroides with the diameter of about 5mm, wherein the light intensity is 150 mu mol phoss/(m 2 & s).
Example 2
The nostoc sphaeroides used in this example was collected from Hubei Hefeng Famazhen rice field and purified in this laboratory to obtain purified algal species. Selecting black globular nostoc sphaeroides (phi 5mm) with good elasticity and strong toughness as a matrix, sequentially washing with tap water for 20min, sufficiently washing with sterile water for 10min, irradiating with UVB ultraviolet lamp for 10min, soaking in 75% alcohol solution for 3min, washing with sterile water for 3 times, and making into inoculum.
The first medium was prepared according to the following recipe: : NaNO30.5g/L，Ca(H2PO4)20.24g/L，MgK2(SO4)20.30g/L，NaHCO30.10g/L,FeCl210mg/L，Na2EDTA2.0mg/L，H3BO35.0mg/L，MnCl2·4H2O3.0mg/L，ZnSO4·7H2O 0.16mg/L，CuSO4·5H2O 0.08mg/L，Na2MoO4·2H2O 0.8mg/L，CoCl2·6H2O0.12 mg/L. The culture medium is prepared by using distilled water, and is filtered by a 0.2um microporous filter membrane for later use.
The inoculum was inoculated into the sterilized complete medium and subjected to aerobic cultivation under 30. mu. mol photons/(m 2. multidot.s) fluorescent lamp at 25 ℃ for 24 h.
After 28 days, obtaining a population with the diameter of 1mm, and transferring the population into a 40L cylindrical photobioreactor for culture according to the ratio of 1: 10; the culture medium used is a first induction culture medium, and comprises the following components: FeCl210mg/L，Na2EDTA2.4mg/L，H3BO33mg/L，MnCl2·4H2O 1.50mg/L，ZnSO4·7H2O 0.16mg/L，CuSO4·5H2O 0.08mg/L，Na2MoO4·2H2O0.4mg/L，CoCl2·6H2O0.04 mg/L. Open cultures were carried out at 50. mu. mol phos/(m 2. multidot.s) at 25 ℃ under 24h light.
After 14 days, the diameter of the nostoc sphaeroides reaches 2.0mm, and the nostoc sphaeroides is transferred to 20m2Culturing in a runway culture pond, wherein the used culture medium is a second induction culture medium and comprises the following components: FeCl210mg/L，Na2EDTA2.4mg/L，H3BO33mg/L，MnCl2·4H2O 1.50mg/L，CuSO4·5H2O 0.08mg/L，CoCl2·6H2O0.04 mg/L. Adopting sunlight as light source, shading with shading net with shading rate of 70% for the first 7 days, shading with shading net with shading rate of 30% for the second 7 days, and culturing for 60 days to obtain golden yellow nostoc sphaeroides with diameter of about 5 mm.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (5)
1. The production method of the golden yellow nostoc sphaeroides is characterized by comprising the following steps of:
1) seed production: selecting nostoc sphaeroids kutz matrix, sterilizing to prepare an inoculum, and transferring the inoculum into a sterilized first culture medium for aeration culture;
2) amplification culture: when the diameter of the nostoc sphaeroides is 0.5-1.0 mm, transferring the nostoc sphaeroides culture in the step 1) into a culture container with a first induction culture medium for open culture according to the proportion of the nostoc sphaeroides culture in the step 1) to the first induction culture medium, wherein the first induction culture medium is 1: 5-30;
3) induction: transferring nostoc sphaeroides with the diameter of 1.5-2.5 mm to a second induction culture medium, and culturing for 30-60 days by illumination to obtain nostoc sphaeroides with the diameter of 4-6 mm;
in the step 1), the aeration culture conditions are as follows:
i) uses air as raw material to increase its CO2The content is 0.1-5% (volume ratio), and the air is used as ventilation gas after filtration and sterilization;
ii) the culture temperature is 20-30 ℃;
iii) light supply is carried out for 24 hours in the culture photoperiod, and the light intensity is 20-50 mu molphosins/(m)2S) using a light source selected from one or more of fluorescent lamps, LED lamps, plant growth lamps or sunlight;
in the step 1), the first culture medium is composed of the following raw materials in types and dosage:
the water is selected from one or more of purified water, single distilled water, tap water, natural surface water and underground water;
in step 2), the composition and dosage of the first induction medium are as follows: FeCl23～20mg/L，Na2EDTA 1～10mg/L，H3BO31～10mg/L，MnCl2·4H2O 0.5～5mg/L，ZnSO4·7H2O 0.1～0.6mg/L，CuSO4·5H2O 0.02～0.2mg/L，Na2MoO4·2H2O 0.01～0.8mg/L，CoCl2·6H2O 0.02～4mg/L；
In step 3), the composition and dosage of the second induction medium are as follows: FeCl23～20mg/L，Na2EDTA 1～10mg/L，H3BO31～10mg/L，MnCl2·4H2O 0.5～5mg/L，CuSO4·5H2O 0.02～0.2mg/L，CoCl2·6H2O 0.02～4mg/L；
In the step 3), the illumination culture is carried out by controlling the light intensity to be more than 100 mu mol phosns/(m)2S) or controlling the wavelength of light to be 400 to 550 nm.
2. The method as claimed in claim 1, wherein the nostoc sphaeroides as a precursor in the step 1) meets the following characteristics:
i) the shape is a regular sphere with the diameter of 3-5 mm, the dry matter content is 1-5 wt%, and the color is dark green;
ii) the steel plate falls from the height of 50cm through a free-fall type, does not crack after being impacted with a hard horizontal plane, and rebounds to the height of 3-20 cm;
iii) the ability to pull the filaments out when broken.
3. The method as claimed in claim 1, wherein the step 1) is performed by one or more of UV sterilization, 75% (by volume) alcohol soaking sterilization, and 0.1-1% (by weight) sodium hypochlorite solution soaking sterilization.
4. The method of claim 1, wherein the first medium is sterilized in step 1) by one or both of filtration sterilization through a 0.2 μm pore size filter and autoclaving.
5. The method according to any one of claims 1 to 4, wherein the nostoc sphaeroides is selected from the nostoc sphaeroides in step 1).
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