CN116724892A - Selenium-enriched nostoc sphaeroids kutz culture method - Google Patents
Selenium-enriched nostoc sphaeroids kutz culture method Download PDFInfo
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- PMYDPQQPEAYXKD-UHFFFAOYSA-N 3-hydroxy-n-naphthalen-2-ylnaphthalene-2-carboxamide Chemical compound C1=CC=CC2=CC(NC(=O)C3=CC4=CC=CC=C4C=C3O)=CC=C21 PMYDPQQPEAYXKD-UHFFFAOYSA-N 0.000 abstract description 9
- 229960001881 sodium selenate Drugs 0.000 abstract description 9
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- 229910052698 phosphorus Inorganic materials 0.000 abstract description 8
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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Classifications
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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
- A01H4/002—Culture media 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 discloses a cultivation method of selenium-enriched nostoc sphaeroids kutz, and belongs to the technical field of biology. According to the invention, the full-nutrition culture medium is prepared by using the selenium-rich hot spring water, so that the selenium source in the selenium-rich hot spring water is fully utilized, a method of adding sodium selenate or sodium selenite exogenously is abandoned, and the health risk existing in the addition of sodium selenate or sodium selenite is avoided; and adding disodium glycerophosphate into the culture medium to provide phosphorus nutrition for nostoc sphaeroids kutz which is easier to absorb, promote nitrogen fixation activity of nostoc sphaeroids kutz, stimulate the nostoc sphaeroids kutz to absorb other nutrient elements, improve growth and metabolic activity of nostoc sphaeroids kutz, and enable nostoc sphaeroids kutz to rapidly produce seeds in a sprouting mode; meanwhile, the invention also increases the selenium absorption capacity of nostoc sphaeroids kutz for a long time under the condition of not affecting the growth and morphological development of nostoc sphaeroids kutz by sulfur-free induction and low-sulfur culture, and has the advantages of simple operation, good safety and low cost.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cultivation method of selenium-enriched nostoc sphaeroids kutz.
Background
Nostoc sphaeroids kutz, the academic name of which is nostoc sphaeroids kutz (Nostoc Sphaeroides), is an edible nitrogen-fixing blue algae with higher nutritive value in rice fields. Nostoc sphaeroids kutz is rich in proteins, fatty acids, minerals and vitamins. Meanwhile, polysaccharide, phycobiliprotein and some lipid substances in nostoc sphaeroids have the functions of resisting oxidization, resisting inflammation, regulating immunity and reducing cholesterol content. Besides the edible value, nostoc sphaeroids kutz plays a great role in nitrogen input in farmlands. Qin et al (2013) found that rice field cultured nostoc sphaeroids kutz was able to significantly increase the nitrogen content (J.appl. Phycol.2013, 25:1737-1745) in the soil surface (0-5 cm) and subsurface (5-10 cm). Researchers such as Liu Yongding in China perform scientific research on artificial breeding of nostoc sphaeroids kutz in the 90 th century and succeed in achieving the aim. Since wild nostoc resources are increasingly reduced, the nostoc circulated in the market at present is mainly artificially cultured nostoc.
Selenium (Se) is one of microelements necessary for human body, and has important physiological effects of lowering blood pressure, regulating immunity, preventing and resisting cancer, etc. However, selenium is not uniformly distributed in China, and a human body cannot synthesize selenium and needs to obtain the selenium from food, so that about 2/3 of the area and about 7 hundred million people in China lack selenium, and 22 provinces and 72% of counties are in a selenium-deficiency state. Selenium intake is deficient for a long period of time, which causes a series of physiological diseases such as keshan disease and large bone joint disease. Selenium can also antagonize other heavy metal elements, and the selenium-enriched food can not only strengthen physique, achieve the purpose of preventing diseases, but also prevent human heavy metal element poisoning. Selenium exists in nature in two forms: inorganic selenium and organic selenium, wherein the inorganic selenium generally refers to sodium selenite and sodium selenate, is obtained from byproducts of metal mineral reservoirs, has high toxicity, is not easy to be absorbed, and is not suitable for people and animals; bioactive in the human body is organic selenium, which exists mostly in the form of selenoamino acids in the organism. Therefore, the selenium-rich organic product has important significance for improving the health condition of people with insufficient selenium intake in China.
In recent years, the development of selenium-enriched nostoc has become an important development direction of nostoc industry, because the protein content in nostoc is high, the amino acid variety of the composition protein is rich, and a material basis is provided for selenium-enriched culture. Chinese patent document CN109392693A discloses a method for artificially culturing selenium-enriched nostoc sphaeroids kutz, wherein sodium selenite is added into a nostoc sphaeroids kutz culture medium to obtain the selenium-enriched nostoc sphaeroids kutz, the selenium content is 350ppm-2000ppm, the diameter of the nostoc sphaeroids kutz reaches 0.8-1.4cm, the protein content reaches over 52 percent, and the nutrition value is rich; although the selenium content in nostoc sphaeroids kutz obtained by the method is high, the adopted selenium source is sodium selenite, which is a highly toxic reagent and can be hardly accepted by consumers; chinese patent document CN109355230A discloses a selenium-enriched culture technique of nostoc sphaeroids kutz, sodium selenite is added into an improved BG-110 liquid culture medium for producing algae species by selenium enrichment of nostoc sphaeroids kutz, so that the final concentration of selenium reaches 1-30mg/L, and the selenium-enriched culture method still has the problem that the selenium-enriched nostoc sphaeroids kutz is obtained by adding extremely toxic sodium selenite and is difficult to be accepted by consumers; chinese patent document CN110257308A discloses a cultivation method of nostoc sphaeroids kutz rich in organic selenium, which comprises the steps of cultivating cordyceps militaris strains into cordyceps militaris fruiting bodies rich in selenium by using a selenium-rich solid medium, obtaining selenium-rich stock solution rich in seleno-amino acid by adopting an enzymolysis method, and cultivating nostoc sphaeroids kutz by using the selenium-rich stock solution to obtain nostoc sphaeroids kutz rich in selenium.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a cultivation method of selenium-enriched nostoc sphaeroids kutz, which uses selenium-enriched hot spring water as a selenium source, and abandons the method of adding sodium selenate or sodium selenite from an external source, thereby avoiding the health risk existing in the addition of sodium selenate or sodium selenite; through regulating the physiological state of nostoc sphaeroids kutz, the nostoc sphaeroids kutz can be in a state of easily absorbing and assimilating selenium in hot spring water. The method has the advantages of good safety, low cost and easy acceptance and acceptance by consumers.
The invention provides a cultivation method of selenium-enriched nostoc sphaeroids kutz, which comprises the following steps:
s1, preparing a nostoc sphaeroids kutz total nutrient medium by using selenium-containing hot spring water for standby;
s2, washing, disinfecting and homogenizing spherical nostoc sphaeroids kutz groups with water to obtain filamentous algae slurry, then coating the filamentous algae slurry on the surface of the total nutrient solid culture medium obtained in the step S1, controlling the temperature and the illumination intensity, and culturing to obtain first-stage nostoc sphaeroids kutz seeds;
s3, transferring the nostoc primary seeds into a full-nutrition liquid culture medium prepared by selenium-rich hot spring water, and carrying out illumination ventilation culture to obtain nostoc secondary seeds;
s4, transferring the nostoc sphaeroids kutz secondary seeds obtained in the step S3 into a sulfur-free culture medium, and performing sulfur starvation culture to obtain a sulfur starvation culture;
s5, transferring the sulfur starvation culture obtained in the step S4 into a low-sulfur culture medium, and culturing under illumination and ventilation to obtain the selenium-enriched nostoc sphaeroids kutz.
Further, the selenium content in the selenium-containing hot spring water in the step S1 is 5-8 mug/L.
Further, the water quality characteristics of the selenium-rich hot spring water in the step S1 are as follows: selenium 7.8 mug/L, na + 15mg/L,K + 1.1mg/L，Mg 2+ 0.8mg/L，Ca 2+ 7.3mg/L，H 2 SiO 3 56mg/L，Cl - 5.7mg/L，SO 4 2- 1.2mg/L，CO 3 2- 46mg/L，F - 8.0mg/L，pH 7.3。
Further, the formula of the total nutrient medium in the step S1 is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 60mg/L，FeCl 3 2mg/L，Na 2 EDTA5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L and CuSO 4 0.02mg/L。
According to the invention, the disodium glycerophosphate is added into the formula of the total nutrient medium, so that phosphorus nutrients which are easier to absorb are provided for nostoc sphaeroids kutz, and the nitrogen fixation activity of nostoc sphaeroids kutz is promoted by using the stoichiometric characteristic of nostoc sphaeroids kutz biosynthesis, the absorption of nostoc sphaeroids kutz to other nutrient elements is stimulated, the growth and metabolic activity of nostoc sphaeroids kutz are improved, and the nostoc sphaeroids kutz can rapidly produce seeds in a sprouting mode.
Further, the total nutrient medium in the step S1 is sterilized by high-pressure steam at 121 ℃ for 30 minutes, and after sterilization, the total nutrient medium is poured into a culture dish before solidification of the medium, and is subjected to static solidification, so that the total nutrient solid medium can be obtained.
Further, the second-level nostoc sphaeroids kutz seeds in the step S4 are obtained by collecting nostoc sphaeroids kutz spheres with the diameter of more than or equal to 3mm through an 8-mesh stainless steel screen disc, and then transferring the nostoc sphaeroids kutz spheres into a sulfur-free culture medium.
Further, the formula of the sulfur-free culture medium in the step S4 is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，CoCl 2 1mg/L，MnCl 2 2mg/L，ZnC l2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuCl 2 0.02mg/L。
According to the invention, the sulfur element nutrient in the culture medium is deleted by setting the sulfur-free culture medium formula, and sulfur deficiency stress is caused on nostoc sphaeroids kutz by short-time sulfur starvation, so that the activity of related enzymes of sulfur and selenium element is stimulated to be absorbed and operated by nostoc sphaeroids kutz, and the uptake capacity of the nostoc sphaeroids kutz for sulfur and selenium is improved.
Further, the sulfur starvation culture time in the step S4 is 3-4 days.
Further, the formula of the low-sulfur culture medium in the step S5 is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 5mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA 5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuSO 4 0.02mg/L。
According to the invention, the formula of the low-sulfur culture medium is arranged, so that sulfur element nutrients in the culture medium are reduced, and the selenium absorption capacity of nostoc sphaeroids kutz is increased for a long time under the condition that the growth and morphological development of nostoc sphaeroids kutz are not influenced.
Further, the diameter of nostoc harvested in the step S5 is 5-8mm.
Compared with the prior art, the invention has the beneficial effects that:
1. in the cultivation method of the selenium-enriched nostoc sphaeroids kutz, the selenium-enriched hot spring water is utilized to prepare the full-nutrition culture medium, the selenium source in the selenium-enriched hot spring water is fully utilized, the method of adding sodium selenate or sodium selenite exogenously is abandoned, the health risk existing in the addition of sodium selenate or sodium selenite is avoided, the operation is simple, the safety is good, and the cost is low.
2. According to the selenium-enriched nostoc sphaeroids kutz culture method provided by the invention, disodium glycerophosphate is added into the nostoc sphaeroids kutz culture medium, so that phosphorus nutrients which are easier to absorb are provided for nostoc sphaeroids kutz, the nitrogen fixation activity of nostoc sphaeroids kutz is promoted by glycerophosphate by utilizing the chemometric characteristic of nostoc sphaeroids kutz biosynthesis, the absorption of nostoc sphaeroids kutz to other nutrient elements is stimulated, the growth and metabolic activity of nostoc sphaeroids kutz are improved, seeds can be rapidly produced in a sprouting mode of nostoc sphaeroids kutz, and nostoc sphaeroids kutz culture is promoted.
3. The cultivation method of the selenium-enriched nostoc sphaeroids kutz provided by the invention constructs a sulfur-free induction culture medium formula and a low-sulfur culture medium formula on the basis of sulfur and selenium co-metabolism to improve the selenium absorption capacity of nostoc sphaeroids kutz, realizes sulfur starvation for 3 days in a short time, improves the sulfur and selenium uptake capacity of nostoc sphaeroids kutz, and also increases the selenium absorption capacity of nostoc sphaeroids kutz for a long time under the condition of not affecting the growth and morphological development of nostoc sphaeroids kutz.
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
The present invention will be further described in detail with reference to the following embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.
The selenium-containing hot spring water used in the embodiment and the comparative example is taken from mountain water hot spring nursing homes in geological bureau of Jiangxi province, and the water quality is characterized in that: selenium 7.8 mug/L, na + 15mg/L,K + 1.1mg/L，Mg 2+ 0.8mg/L，Ca 2+ 7.3mg/L，H 2 SiO 3 56mg/L，Cl - 5.7mg/L，SO 4 2- 1.2mg/L，CO 3 2- 46mg/L，F - 8.0mg/L，pH 7.3。
EXAMPLE 1 cultivation of selenium-enriched nostoc sphaeroids kutz of the present invention
The cultivation method of the selenium-enriched nostoc sphaeroids kutz of the embodiment comprises the following steps:
s1, preparing a nostoc sphaeroids kutz total nutrient medium by using hot spring water (taken from mountain water hot spring nursing homes of geological bureau in Jiangxi province) with the selenium content of 5-8 mug/L; the nostoc sphaeroids kutz total nutrient medium formula is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 60mg/L，FeCl 3 2mg/L，Na 2 EDTA5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuSO 4 Sterilizing the culture medium formula materials at 121 ℃ for 30 minutes by high-pressure steam at 0.02mg/L and 2% of agar, pouring the culture medium formula materials into a culture dish before solidification of the culture medium after sterilization, and standing for solidification to obtain a full-nutrition solid culture medium for later use;
s2, washing spherical nostoc sphaeroids kutz groups with water, sterilizing the surfaces of the spherical nostoc sphaeroids kutz groups with ethanol with the volume concentration of 75%, washing the spherical nostoc sphaeroids kutz groups with sterile water for 3 times, homogenizing the nostoc sphaeroids kutz groups to obtain filamentous algae pulp, coating the spherical nostoc sphaeroids kutz pulp on the surfaces of the total nutrient solid culture medium obtained in the step S1, and carrying out illumination intensity of 30 mu mol m at the temperature of 25 DEG C -2 ·s -1 Obtaining first-class seeds of nostoc sphaeroids kutz;
s3, transferring the nostoc primary seeds into a full-nutrition liquid culture medium, and carrying out illumination ventilation culture in a 5L glass tank until the diameter of the nostoc is about 3-4mm to obtain nostoc secondary seeds;
s4, collecting secondary seed spheres of nostoc sphaeroids kutz which are larger than or equal to 3mm by using an 8-mesh stainless steel screen disc, transferring the secondary seed spheres into a sulfur-free culture medium prepared by selenium-rich hot spring water, and performing sulfur starvation culture for 3 days to obtain a sulfur starvation culture; wherein the formula of the sulfur-free culture medium is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA 5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，CoCl 2 1mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuCl 2 0.02mg/L；
S5, transferring the sulfur starvation culture obtained in the step S4 into a low-sulfur culture medium, and culturing by illumination and ventilation until the diameter of nostoc sphaeroids kutz is about 5-8mm, thereby obtaining the selenium-enriched nostoc sphaeroids kutz of the embodiment; wherein the formula of the low-sulfur culture medium is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 5mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA 5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuSO 4 0.02mg/L。
EXAMPLE 2 cultivation of selenium-enriched nostoc sphaeroids kutz of the present invention
The cultivation method of the selenium-enriched nostoc sphaeroids kutz of the embodiment comprises the following steps:
s1, preparing a full-nutrition culture medium by using hot spring water (taken from mountain and water hot spring nursing homes of geological bureau of Jiangxi province) with the selenium content of 5-8 mug/L; wherein the formula of the total nutrient medium is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 60mg/L，FeCl 3 2mg/L，Na 2 EDTA 5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuSO 4 Sterilizing the culture medium formula materials at 121 ℃ for 30 minutes by high-pressure steam at 0.02mg/L and 2% of agar, pouring the culture medium formula materials into a culture dish before solidification of the culture medium after sterilization, and standing for solidification to obtain a full-nutrition solid culture medium for later use;
s2, washing spherical nostoc sphaeroids kutz groups with water, disinfecting the surfaces of the spherical nostoc sphaeroids kutz groups with 75% ethanol, washing the spherical nostoc sphaeroids kutz groups with sterile water for 3 times, homogenizing the nostoc sphaeroids kutz groups to obtain filamentous algae pulp, and then homogenizing the nostoc sphaeroids kutz pulpCoating on the surface of the total nutrient solid culture medium obtained in the step S1, and controlling the temperature to 27.5 ℃ and the illumination intensity to 45 mu mol.m -2 ·s -1 Obtaining first-class seeds of nostoc sphaeroids kutz;
s3, transferring the nostoc primary seeds into a full-nutrition liquid culture medium, and carrying out illumination ventilation culture in a 5L glass tank until the diameter of the nostoc is about 3-4mm to obtain nostoc secondary seeds;
s4, collecting secondary seed spheres of nostoc sphaeroids kutz which are larger than or equal to 3mm by using an 8-mesh stainless steel screen disc, transferring the secondary seed spheres into a sulfur-free culture medium prepared by selenium-rich hot spring water, and performing sulfur starvation culture for 3 days to obtain a sulfur starvation culture; wherein the formula of the sulfur-free culture medium is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA 5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，CoCl 2 1mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuCl 2 0.02mg/L；
S5, transferring the sulfur starvation culture obtained in the step S4 into a low-sulfur culture medium prepared by selenium-rich hot spring water, and carrying out illumination ventilation culture until the diameter of nostoc sphaeroids kutz is about 5-8mm, thereby obtaining the selenium-rich nostoc sphaeroids kutz of the embodiment; wherein the formula of the low-sulfur culture medium is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 5mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA 5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuSO 4 0.02mg/L。
EXAMPLE 3 cultivation of selenium-enriched nostoc sphaeroids kutz of the present invention
The cultivation method of the selenium-enriched nostoc sphaeroids kutz of the embodiment comprises the following steps:
s1, preparing a full-nutrition culture medium by using hot spring water (taken from mountain and water hot spring nursing homes of geological bureau of Jiangxi province) with the selenium content of 5-8 mug/L; wherein the whole nutritionThe formula of the culture medium is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 60mg/L，FeCl 3 2mg/L，Na 2 EDTA 5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuSO 4 Sterilizing the culture medium formula materials at 121 ℃ for 30 minutes by high-pressure steam at 0.02mg/L and 2% of agar, pouring the culture medium formula materials into a culture dish before solidification of the culture medium after sterilization, and standing for solidification to obtain a full-nutrition solid culture medium for later use;
s2, washing spherical nostoc sphaeroids kutz colony with water, sterilizing with 75% ethanol surface, washing with sterile water for 3 times, homogenizing nostoc sphaeroids kutz to obtain filamentous algae slurry, and coating the nostoc sphaeroids kutz slurry on the surface of the total nutrient solid culture medium obtained in the step S1 at the temperature of 30 ℃ and the illumination intensity of 60 mu mol m -2 ·s -1 Obtaining first-class seeds of nostoc sphaeroids kutz;
s3, transferring the nostoc primary seeds into a full-nutrition liquid culture medium, and carrying out illumination ventilation culture in a 5L glass tank until the diameter of the nostoc is about 3-4mm to obtain nostoc secondary seeds;
s4, collecting secondary seed spheres of nostoc sphaeroids kutz which are larger than or equal to 3mm by using an 8-mesh stainless steel screen disc, transferring the secondary seed spheres into a sulfur-free culture medium prepared by selenium-rich hot spring water, and performing sulfur starvation culture for 3 days to obtain a sulfur starvation culture; wherein the formula of the sulfur-free culture medium is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，CoCl 2 1mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuCl 2 0.02mg/L；
S5, transferring the sulfur starvation culture obtained in the step S4 into a low sulfur culture medium prepared by selenium-rich hot spring water, and culturing under illumination and ventilation until the diameter of nostoc sphaeroids kutz is about 5-8mm to obtain the preparation methodIs rich in selenium nostoc; wherein the formula of the low-sulfur culture medium is as follows: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 5mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuSO 4 0.02mg/L。
Comparative example 1
The difference between this comparative example and example 1 is that: the primary and secondary seeds of nostoc sphaeroids kutz of the present comparative example were cultured with BG110 medium prepared with hot spring water, and the sulfur starvation medium culture and the low sulfur medium culture of steps S4 and S5 of the present comparative example were replaced with BG110 medium. The formula of the BG110 medium is disclosed in the reference Rippka R.et al 1979, J.Gen.Microbiol.11:1-61 (https:// doi.org/10.1099/00221287-111-1-1), and the rest steps and parameters are exactly the same as in example 1.
Comparative example 2
The difference between this comparative example and example 1 is that: the formula of the total nutrient medium of the comparative example does not contain 1mg/L of disodium glycerophosphate.
Comparative example 3
The difference between this comparative example and example 1 is that: in step S4 of the comparative example, the non-sulfur starvation medium culture was used instead of the sulfur starvation culture, that is, the total nutrient medium culture prepared in step S1 was continued, and the other steps and parameters were identical to those of example 1.
Comparative example 4
The difference between this comparative example and example 1 is that: after the sulfur starvation culture in step S4 of this comparative example, the culture was continued in step S5 using a sulfur-free medium, and the remaining steps and parameters were exactly the same as in example 1.
Comparative example 5
The difference between this comparative example and example 1 is that: in step S5 of this comparative example, the culture was continued by replacing the low-sulfur medium with the total nutrient medium culture prepared in step S1, and the remaining steps and parameters were identical to those of example 1.
Test example 1 examination of the excitation Effect of disodium glycerophosphate on growth and metabolism of nostoc sphaeroids kutz
1. Test sample: secondary seeds of nostoc sphaeroids kutz cultivated in examples 1 to 3 and comparative examples 1 to 2;
2. the test method comprises the following steps: detecting nitrogen and phosphorus content, nitrogen enzyme activity and biomass in unit volume in the secondary seed cells of nostoc sphaeroids kutz;
3. test results:
TABLE 1 intracellular phosphorus, nitrogen, and Nitrogen fixing enzyme Activity and biomass of Nostoc sphaeroids kutz
As is clear from Table 1, the nitrogen and phosphorus content, the activity of the azotase and the biomass in unit volume of the secondary seeds of nostoc sphaeroids kutz cultivated in examples 1-3 are higher than those of the secondary seeds of nostoc sphaeroids kutz cultivated in comparative examples 1-2, which shows that the addition of disodium glycerophosphate to the culture medium provides phosphorus nutrients easier to absorb for nostoc sphaeroids kutz, and the chemometric characteristic of the biosynthesis of nostoc sphaeroids kutz is utilized to promote the nitrogen fixation activity of nostoc sphaeroids kutz, so that the absorption of nostoc sphaeroids kutz to other nutrient elements is stimulated, the growth and metabolic activity of nostoc sphaeroids kutz are improved, and the phosphorus, nitrogen, the activity of the azotase and the biomass in nostoc sphaeroids kutz cells are improved. The culture medium used in comparative examples 1-2 did not contain disodium glycerophosphate, and all affected the absorption of other nutrient elements by nostoc sphaeroids kutz.
Test example 2 stimulation effect of Sulfur starvation on selenium absorption by nostoc sphaeroids kutz
1. Test sample: examples 1-3 and comparative example 3 nostoc sphaeroids kutz cultured in step S4;
2. the test method comprises the following steps: the affinity of nostoc for selenium absorption (expressed as the Km value of the michaelis equation constant) was tested, with lower Km values representing higher affinities for selenium;
3. test results:
TABLE 2 affinity of nostoc for selenium uptake
| Km value (mu M) | |
| Example 1 | 8.17 |
| Example 2 | 8.13 |
| Example 3 | 8.16 |
| Comparative example 3 | 41.69 |
As is clear from the results of Table 2, km values of the nostoc sphaeroids kutz subjected to sulfur starvation treatment in examples 1-3 of the present invention are 8.15-8.17 mu M, and Km values of the nostoc sphaeroids kutz comparative example 3 are 41.69 mu M, which indicates that the sulfur and selenium uptake capacity of nostoc sphaeroids kutz is improved by setting a sulfur-free medium formulation, deleting sulfur element nutrients in the medium, and sulfur-deficiency stress on nostoc sphaeroids kutz caused by short-time 3-day sulfur starvation culture, and activating activities of enzymes related to absorption, operation of sulfur and selenium elements of nostoc sphaeroids kutz.
Test example 3 selenium-enriched nostoc sphaeroids kutz culture method and selenium-enriched effect investigation
1. Test sample: nostoc sphaeroids kutz cultivated in examples 1 to 3 and comparative examples 1 to 5;
2. the test method comprises the following steps: sending nostoc sphaeroids kutz sample to third party radio and television measurement (Hunan) limited company to detect the selenium content in nostoc sphaeroids kutz, and detecting according to food safety national standard of measuring selenium in food GB 5009.93-2017;
3. test results:
TABLE 3 selenium content in nostoc sphaeroids kutz
As is clear from Table 3, the selenium content of the nostoc sphaeroids kutz cultivated in examples 1-3 of the present invention is up to 0.79-0.81mg/Kg, while the selenium content of the nostoc sphaeroids kutz cultivated in comparative examples 1-3 and 5 is only 0.21-0.45mg/Kg, the nostoc sphaeroids kutz of comparative example 4 is cracked in step S5, and spherical nostoc sphaeroids kutz of 5-8mm cannot be cultivated successfully, which means that the present invention fully uses the selenium source in the selenium-rich spring water, adopts a brand-new culture medium formulation containing disodium glycerophosphate, combines sulfur starvation and low sulfur culture medium cultivation, can increase the absorption capacity of the nostoc sphaeroids kutz to selenium for a long time without affecting the growth and morphological development of the nostoc sphaeroids kutz, abandons the method of adding sodium selenate or sodium selenite, avoids the health risk existing in the addition of sodium selenate or sodium selenite, and has better safety.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A cultivation method of selenium-enriched nostoc sphaeroids kutz is characterized by comprising the following steps:
s1, preparing a nostoc sphaeroids kutz total nutrient medium by using selenium-containing hot spring water for standby;
s2, washing, disinfecting and homogenizing spherical nostoc sphaeroids kutz groups with water to obtain filamentous algae slurry, then coating the filamentous algae slurry on the surface of the total nutrient solid culture medium obtained in the step S1, controlling the temperature and the illumination intensity, and culturing to obtain first-stage nostoc sphaeroids kutz seeds;
s3, transferring the nostoc sphaeroids kutz first-stage seeds into a full-nutrition liquid culture medium, and carrying out illumination ventilation culture to obtain nostoc sphaeroids kutz second-stage seeds;
s4, transferring the nostoc sphaeroids kutz secondary seeds obtained in the step S3 into a sulfur-free culture medium, and performing sulfur starvation culture to obtain a sulfur starvation culture;
s5, transferring the sulfur starvation culture obtained in the step S4 into a low-sulfur culture medium, and culturing under illumination and ventilation to obtain the selenium-enriched nostoc sphaeroids kutz.
2. The method for culturing selenium-enriched nostoc sphaeroids kutz according to claim 1, wherein the selenium content in the selenium-containing hot spring water in the step S1 is 5-8 μg/L.
3. The cultivation method of selenium-enriched nostoc sphaeroids kutz according to claim 1, wherein the water quality characteristics of the selenium-enriched hot spring water in the step S1 are as follows: selenium 7.8 mug/L, na + 15mg/L,K + 1.1mg/L，Mg 2+ 0.8mg/L，Ca 2+ 7.3mg/L，H 2 SiO 3 56mg/L，Cl - 5.7mg/L，SO 4 2- 1.2mg/L，CO 3 2- 46mg/L，F - 8.0mg/L，pH 7.3。
4. The method for culturing selenium-enriched nostoc sphaeroids kutz according to claim 1, wherein the formula of the total nutrient medium of the step S1 is: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 60mg/L，FeCl 3 2mg/L，Na 2 EDTA5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L and CuSO 4 0.02mg/L。
5. The method for culturing selenium-enriched nostoc sphaeroids kutz according to claim 1, wherein the total nutrient medium of the step S1 is sterilized by high-pressure steam at 121 ℃ for 30 minutes, and after sterilization, poured into a culture dish before the medium is solidified, and is solidified at rest, thus obtaining the total nutrient solid medium.
6. The method according to claim 1, wherein the second seed of nostoc sphaeroids kutz in step S4 is obtained by collecting nostoc sphaeroids kutz spheres of 3mm or more through a 8-mesh stainless steel screen plate before transferring them to a sulfur-free medium.
7. The method for culturing selenium-enriched nostoc sphaeroids kutz according to claim 1, wherein the sulfur-free medium formulation in the step S4 is: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，CoCl 2 1mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuCl 2 0.02mg/L。
8. The cultivation method of selenium-enriched nostoc sphaeroids kutz according to claim 1, wherein the sulfur starvation cultivation time in the step S4 is 3 to 4 days.
9. The method for culturing selenium-enriched nostoc according to claim 1, wherein the formula of the low-sulfur medium in step S5 is: disodium glycerophosphate 1mg/L, naH 2 PO 4 30mg/L，MgSO 4 5mg/L，MgCl 2 40mg/L，FeCl 3 2mg/L，Na 2 EDTA5mg/L，CaCl 2 50mg/L，K 2 CO 3 50mg/L，MnCl 2 2mg/L，ZnCl 2 0.5mg/L，NaMoO 4 0.7mg/L，H 3 BO 3 5mg/L，CuSO 4 0.02mg/L。
10. The cultivation method of selenium-enriched nostoc sphaeroids kutz according to claim 1, wherein the diameter of nostoc sphaeroids kutz harvested in step S5 is 5-8mm.
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