CN116904318B - Method for stably proliferating noctilucent algae based on mixed culture - Google Patents

Method for stably proliferating noctilucent algae based on mixed culture Download PDF

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CN116904318B
CN116904318B CN202310975865.9A CN202310975865A CN116904318B CN 116904318 B CN116904318 B CN 116904318B CN 202310975865 A CN202310975865 A CN 202310975865A CN 116904318 B CN116904318 B CN 116904318B
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姬南京
王君玥
申欣
毛宁
陈奕帆
蔡月凤
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Abstract

The invention discloses a method for stably proliferating noctilucent algae based on mixed culture, which is characterized in that noctilucent algae (Noctiluca scintillans) and heterodera stupeutica (Heterocapsa steinii) are co-cultured, and a co-culture system contains nutrient salts, wherein the nutrient salts comprise N element, si element, P element, trace metal element and vitamin. The method is simple to operate, and the level of nutrient salt does not need to be strictly controlled; the noctilucent algae grows rapidly in a co-culture system with the noctilucent algae, the high-density and high-growth-rate noctilucent algae has no inhibition effect on the growth of the noctilucent algae, the noctilucent algae population density increases along with the increase of the noctilucent algae population density and the nutrient salt concentration, and the noctilucent algae population density can reach (109+/-3.9) cells/mL within 6 days.

Description

Method for stably proliferating noctilucent algae based on mixed culture
Technical Field
The invention relates to a method for stably proliferating noctilucent algae based on mixed culture, belonging to the field of phytoplankton culture.
Background
Red tide generally refers to the phenomenon that some marine microalgae, protozoa or bacteria excessively multiply or aggregate in a water body to cause the water body to change color. In recent years, with the aggravation of water eutrophication, global climate change, ocean acidification and other phenomena, the scale and frequency of red tide generation generally have a remarkable increasing trend. Noctiluca (Noctiluca scintillans) belongs to the phylum dinoflagellate and as one of the typical red tide organisms, large-area red tides have been formed in various sea areas around the world. In addition, noctilucent algae are also receiving social attention because they form a "blue fluorescent sea". Noctilucent algae include two types: red noctiluca and green noctiluca. The red noctilucent algae is pure heterotrophic algae, and can only obtain nutrition for growth and propagation by ingestion of phytoplankton such as diatoms, bacteria, copepod eggs and the like, and is distributed in coastal areas of China. In view of the high public concern about red tide generation of noctiluca, many scholars have developed red noctiluca related research in recent years. In addition, the "blue tear" phenomenon is of great concern, and the public fills the mystery in this ecological phenomenon, and the underlying cause is poorly understood.
The red noctilucent algae is cultivated by adding bait algae liquid, and the growth of the noctilucent algae can be influenced by factors such as the selection, the addition amount and the addition of nutrient salts of the bait algae, for example, the death of the noctilucent algae can be caused by excessive addition of some bait or nutrient salts, so that the laboratory noctilucent algae is cultivated in a small volume, the experimental requirements can not be met at times, and the bait is continuously fed and the requirement on the addition amount of the bait or the nutrient salts is higher. Thus, the factors bring a plurality of problems to experiments, and limit the large-volume culture and research of the noctilucent algae under laboratory conditions.
The patent application (application number: 202111357413.1, name: a noctilucent algae rapid proliferation culture method) discloses a noctilucent algae rapid proliferation culture method, which is a method for obtaining high-density noctilucent algae in a short time at one time through a chain of inorganic nutrition-bait algae-noctilucent algae by providing low-level nutrient salt addition; however, the patent application needs to strictly control the initial density and the cell state of the bait alga Phaeophyta sub-heart, is relatively complex to operate and is easily influenced by the bait alga state and the like, and the nutrient salt concentration in a culture system needs to be strictly controlled in the early stage of culture, if the nutrient salt concentration is too high, a great deal of bait alga is bred, and the noctilucent alga is quickly killed due to the action of chemical sense.
Therefore, there is a need for a method of culturing noctilucent algae that is simple to operate and does not require strict control of nutrient salt concentration.
Disclosure of Invention
The invention aims to: the technical problem to be solved by the invention is to provide a method for stably proliferating noctilucent algae based on mixed culture, which is simple to operate and does not need to strictly control the density and salt concentration of bait algae. Therefore, the invention has no fixed addition amount of the Isodon japonicus, and the volume ratio of the f/2-Si culture medium to the total amount of the algae liquid (the Isodon stonecrop and the noctiluca) is 1 per mill as long as the added Isodon stonecrop is active.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a method for stably proliferating noctiluca based on mixed culture, which is characterized in that noctiluca (Noctiluca scintillans) and xenopus stonecrop (Heterocapsa steinii) are co-cultured, and the co-culture system contains nutrient salts, wherein the nutrient salts comprise N element, si element, P element, trace metal element and vitamin.
Wherein the noctilucent algae comprises red noctilucent algae.
Wherein the substance containing the N element comprises NaNO 3 The Si-containing substance includes Na 2 SiO 3 ·9H 2 O, the substance containing the P element comprises NaH 2 PO 4 ·2H 2 O。
Wherein the trace metal element comprises Fe, cu, zn, co, mn or Mo.
Wherein the material containing Fe comprises FeCl 3 ·6H 2 O, the substance containing Cu comprises CuSO 4 ·5H 2 O, the Zn-containing substance comprises ZnSO 4 ·7H 2 O, the substance containing the Co comprises CoCl 2 ·6H 2 O, the substances containing Mn include MnCl 2 ·4H 2 O, the Mo-containing material including Na 2 MoO 4 ·2H 2 O。
Wherein the vitamins include vitamin B 12 H or B 1 。
Wherein the NaNO 3 The concentration of (2) is 0.551×10 -4 ～8.82×10 -4 mol/L of the Na 2 SiO 3 ·9H 2 The concentration of O is0.663×10 -5 ～1.06×10 -4 mol/L, the NaH 2 PO 4 ·2H 2 O concentration of 0.226×10 -5 ～3.62×10 - 5 mol/L。
Wherein the FeCl 3 ·6H 2 O concentration of 0.731×10 -6 ～1.17×10 -5 mol/L, the CuSO 4 ·5H 2 O concentration of 0.246×10 -8 ～3.93×10 -8 mol/L, the ZnSO 4 ·7H 2 O concentration of 0.478×10 -8 ～7.65×10 -8 mol/L of the CoCl 2 ·6H 2 O concentration of 0.263×10 -8 ～4.2×10 -8 mol/L of MnCl 2 ·4H 2 O concentration of 0.569×10 -7 ～9.1×10 -7 mol/L of the Na 2 MoO 4 ·2H 2 O concentration of 0.163×10 -8 ～2.6×10 - 8 mol/L。
Wherein the vitamin B 12 Is 0.231×10 in concentration -10 ～3.69×10 -10 mol/L, the concentration of vitamin H is 0.128×10 -9 ～2.05×10 -9 mol/L, the concentration of vitamin B1 is 0.185 multiplied by 10 -7 ～2.96×10 -7 mol/L。
The invention successfully screens out a proper bait alga, and automatically establishes a disposable bait casting culture method of noctilucent alga combined with a large-volume culture device. The high-density luminous algae can be cultivated in a large-volume cultivation device for a long time and can be obtained only by adding nutrient salt to allow the luminous algae to grow and reproduce, so that a guarantee is provided for luminous algae research.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
1. the noctilucent algae grows rapidly in a system co-culturing with the anophelus stephensi, the anophelus stephensi with high density and high growth rate has no inhibition effect on the growth of the noctilucent algae, the noctilucent algae population density increases along with the increase of the anophelus stephensi population density and the nutrient salt concentration, and the noctilucent algae population density of the f/2-Si treatment group can reach (109+/-3.9) cells/mL within 6 days;
2. the noctilucent algae in the mixed culture system has a plurality of filled food bubbles, which indicates that the noctilucent algae can ingest the heterodera schneideriana and grow and reproduce by utilizing the nutritional value of the noctilucent algae;
3. only the nutrient salt is added for growth and propagation of the bait algae, so that sufficient bait is provided for the noctilucent algae, the noctilucent algae can be cultivated in a large-volume cultivation device for a long time and high density noctilucent algae can be obtained, and the research of the noctilucent algae is guaranteed;
4. the establishment of the culture method can lead the masses to realize continuous culture of the noctilucent algae under the condition of no laboratory, and understand the real reason behind the natural wonder at the same time of enjoying the noctilucent algae 'blue tears', namely the blue tears are not given away from nature, but are ecological anomaly, so as to realize science popularization knowledge, remind the public to cognize the ocean, care the ocean and protect the ocean.
Drawings
FIG. 1 shows the population dynamics of noctiluca and Isodon japonicus under different nutrient salt conditions: FIG. 1A is a cell density of noctilucent red algae; FIG. 1B is a cell density of Isodon japonicus;
FIG. 2 is a diagram of noctilucent algal cells co-cultured with Isodon stonecrop: FIG. 2A is a diagram of noctilucent algae cells when co-cultured with Isodon stonecrop for 0 h; FIG. 2B is a diagram of noctilucent algae cells when co-cultured with Isodon stonecrop for 6 h;
FIG. 3 shows growth curves of noctilucent algae under large scale culture conditions;
FIG. 4 shows the population dynamics of noctiluca and Alternaria akabane under different nutrient salt conditions: FIG. 4A is a diagram of luminous algae cell density; FIG. 4B is a graph showing cell density of Alternaria akabane;
FIG. 5 shows the population dynamics of Shelving Alternaria alternata and Shelter skeletonema under different nutrient salt conditions: FIG. 5A is a diagram of luminous algae cell density; FIG. 5B is skeletonema costatum cell density;
FIG. 6 is a diagram of noctilucent algal cells co-cultured with different algal species: FIG. 6A is a co-culture with Isodon japonicus; FIG. 6B is a co-culture with Skeletonema costatum; FIG. 6C is a co-culture with Alternaria akabane.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
1. Preparation of the culture medium:
(1) Obtaining seawater: seawater used for culturing red noctilucent algae is obtained from sea area of Lianyuangang, salinity is adjusted to 30+ -1%o, and the sea water is filtered by a filter membrane with pore diameter of 0.22 μm, and then sterilized in a full-automatic sterilizing pot at 121deg.C for 30min, and cooled to room temperature for use.
(2) f/2 configuration of the culture medium: first, double distilled water (ddH) 2 O) preparing 500mL of mother liquor of nitrogen, phosphorus, silicon (Si), trace metal and Vitamin (Vitamin) (table 1), filtering with a 0.22 μm needle filter for later use, and finally adding into sterilized seawater. Wherein the volume of the f/2 culture medium is 1 per mill of the volume of sterilized seawater.
TABLE 1f/2 Medium formulation
(3) f/8 configuration of the culture medium: first, double distilled water (ddH) 2 O) preparing 500mL of mother liquor of nitrogen, phosphorus, silicon (Si), trace metal and Vitamin (Vitamin) (table 2), filtering with a 0.22 μm needle filter for later use, and finally adding into sterilized seawater. Wherein the volume of the f/8 culture medium is 1 per mill of the volume of sterilized seawater.
TABLE 2f/8 Medium formulation
(4) f/32 configuration of the culture medium: first, double distilled water (ddH) 2 O) preparing 500mL of mother liquor of nitrogen, phosphorus, silicon (Si), trace metal and Vitamin (Vitamin) (table 3), filtering with a 0.22 μm needle filter for later use, and finally adding into sterilized seawater. Wherein the volume of the f/32 culture medium is1% by volume of sterilized seawater.
Table 3 f/32 Medium formulation
2. Culture of Isodon stonecrop (Heterocapsa steinii):
(1) Acquisition of the Isoniapis schneid: the Isodon stonecrop is bait algae of red noctilucent algae, and is separated from the coastal area of Jiangsu yun gang. After the water sample is collected, the micro-tube is used for separating the heterodera schneideriana single cells, and the heterodera schneideriana single cells are transferred into an illumination incubator to be cultured by using an f/2-Si culture medium.
(2) Identification of the Isodon stonecrop: the algal strain DNA was extracted using a Amanita Biotechnology (Beijing) Co., ltd. TD433 DNA purification and concentration kit, and PCR amplification was performed on the algal strain sequence using 18Scom-F1 (GCTTGTCTCAAAGATTAAGCCATGC) and 18Scom-R1 (CACCTACGGAAACCTTGTTACGAC) primers. The volume of the PCR amplification system was 50. Mu.L: 25. Mu.L of 2X Accurata Taq Master Mix (dye plus), 4. Mu.L of 18ScomF-3end+18ScomF-3end primer (10. Mu.M), 17. Mu.L of ddH2O. The PCR reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 10s, annealing at 55℃for 30s, extension at 72℃for 1min, and extension at 72℃for 10min after 32 cycles. Sequencing was performed by Shanghai Meimapu biosome, and the measured sequences were aligned in NCBI (National Center for Biotechnology Information) database after correction, splicing and analysis using software such as DNAMAN, chromas and Seq Man. The results show that the similarity of the strain with the Isodon stonecrop (accession number: HQ 902267.1) reaches 100 percent.
(2) Culture of Isodon japonicus: the step of adding the Isodon stonecrop to the prepared seawater at a volume ratio of about 1:25, and adding an f/2-Si culture medium with a total volume of 1%. The culture temperature is 20 ℃, and the illumination intensity is 100+/-10 mu E m -2 s -1 Light-dark period 14: and 10h.
3. Culture of noctilucent red algae (Noctiluca scintillans):
(1) Obtaining red noctilucent algae: isolated from the coastal region of the Jiangsu cloud harbor island. After collecting the surface seawater, absorbing noctilucent alga single cells by using a large-caliber straw, transferring the noctilucent alga single cells into an illumination incubator, and culturing by using sterilized seawater.
(2) Identification of red noctilucent algae: DNA of the noctilucent strain was extracted using a Ambrosia crispa Biotechnology (Beijing) Co., ltd. TD433 DNA purification and concentration kit, and then diluted to 10 ng/. Mu.L was used as a DNA template using 18Scomf-3end (GTCGTAACAAGGTTTCCGTAGGTG) and com28SR 1 (TCACGCATAGTTCACCATCTTTCG) primer, PCR amplification is carried out on the ITS region sequence of the noctiluca. The volume of the PCR amplification system was 50. Mu.L: 25. Mu.L of 2X Accurata Taq Master Mix (dye plus), 4. Mu.L of 18ScomF-3end+18ScomF-3end primer (10. Mu.M), 17. Mu.L of ddH2O. The PCR reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 10s, annealing at 55℃for 30s, extension at 72℃for 1min, and extension at 72℃for 10min after 32 cycles.
The PCR product is subjected to agarose gel electrophoresis, then the target fragment of the PCR product is recovered and purified by using a common agarose DNA recovery kit of the Tiangen DP209-02, and the target fragment is combined with TakarapMD TM After the 19-T Vector Cloning Kit vector ligation, the ligation product was transformed into Beijing full gold biotechnology Co., ltd., trans1-T1 phase resistance chemistry competent cells. 200. Mu.L of the transformed competent cells, 40. Mu.L of X-Gal and 5. Mu.L of IPTG were thoroughly mixed and spread on LB agar medium containing Amp, and cultured overnight in a constant temperature incubator at 37 ℃.
Pure white monoclonal is selected and placed in 1mL of LB liquid medium with Amp, and the culture is carried out for 4 to 6 hours in a constant temperature oscillator at 37 ℃ and 200 rpm. The bacterial liquid was used as a template, and PCR amplification was performed again using 18ScomF-3end and com28SR1 primers, and the volume of the PCR amplification system was 20. Mu.L: 10. Mu.L of 2X Accurata Taq Master Mix (dye plus), 1. Mu.L of DNA template, 1.5. Mu.L of 18ScomF-3end+18ScomF-3end primer (10. Mu.M), 7.5. Mu.L of ddH2O. The PCR reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 10s, annealing at 55℃for 30s, extension at 72℃for 1min, and extension at 72℃for 10min after 32 cycles. After agarose gel electrophoresis of the PCR products, it was observed whether the bands were single and correct. Bacterial solutions corresponding to the bright bands are selected and sent to Shanghai Meimapu biological company for sequencing, and the detected sequences are corrected, spliced and analyzed by using DNAMAN, chromas, seq Man and other software, and then sequence comparison is carried out in a NCBI (National Center for Biotechnology Information) database. The result shows that the similarity of the sequence of the strain and noctilucent algae (accession number: GQ 380592.1) is 98.89%.
(3) The culture method of the red noctilucent algae comprises the following steps: taking the prepared sterilized seawater as a culture medium, adding the Isodon stonecrop and the culture medium into the red noctilucent algae liquid at the same time, and only adding the culture medium every 6-7 days later. Wherein, the red noctilucent algae is mixed into sterilized natural seawater to obtain the red noctilucent algae liquid.
Example 1 population dynamics of noctiluca rubra and Isodon septemlobus under different nutrient salts
Co-culturing red noctilucent algae and the anopheles stephensi in the exponential growth period. And 3 nutrient salt concentrations, namely f/2, f/8 and f/32, are set for the co-culture system, so that an experimental group (f/2-Si group, f/8-Si group and f/32-Si group) is obtained, and the same amount of natural seawater is added as a control group. The experiment used 100mL Erlenmeyer flask, the co-culture volume of each treatment group was 50mL, and the initial cell densities of noctiluca rubra and Isodon schneiderianum were 5cells/mL and 1X 10, respectively 4 The addition amount of the culture medium of each group is 1 per mill of the total volume of the algae liquid of the Isodon stonecrop and the noctiluca, namely 0.05mL; each experimental group was set with 3 biological replicates. The culture temperature is 20 ℃, and the light-dark period is 14h: for 10 hours, the illumination intensity is 100+/-10 mu E.m -2 ·s -1 During which bait is not fed. Cell counts were performed for red noctilucent algae and xenopus stonecrop every 2 d. The results show (FIG. 1), under the condition of 3 kinds of nutritive salts, red luminous algae can grow rapidly in the co-culture system with the Isodon schneiderianum, the growth of the luminous algae is not inhibited by the Isodon schneiderianum with high density and high growth rate, the population density of the red luminous algae of the f/2-Si treatment group increases along with the increase of the population density of the Isodon schneiderianum and the nutritive salt concentration, and the population density of the red luminous algae of the f/2-Si treatment group rises to (10) at the 6d 9 ±3.9)cells/mL。
In addition, the luminous algae in the co-culture system is transferred into a cell culture plate by using a large-caliber straw, and is placed under an inverted microscope for observation and recording. The results show (FIG. 2) that more and more food bubbles exist in red noctilucent algae in the f/2-Si group co-culture system than in noctilucent algae without feeding, indicating that red noctilucent algae can ingest and utilize its nutritional value for growth and reproduction.
Example 2 growth of red noctilucent algae under macroculture conditions
Co-culturing red noctilucent algae and the heterophis stigmariurus. The experiment uses a 25L culture fish tank, the culture volume is 11L, and the initial cell densities of the red noctiluca and the Isodon schneideriana are 19cells/mL and 3×10 respectively 3 The cells/mL co-culture system used standard f/2-Si medium. The cultivation temperature is 20 ℃, the LED lamp of 11000K is used for illumination, and the light-dark period is 14h: and feeding the baits is not carried out in the period of 10 hours. Cells were counted daily for red noctilucent algae. The results show (FIG. 3) that red noctilucent algae grow rapidly when co-cultured with Isodon stonecrop under the condition of large system culture, and the population density of red noctilucent algae rises to 90cells/mL at 9d, which indicates that the method is applicable to the condition of large system culture.
Comparative example population dynamics of red noctilucent algae and three typical bait algae under different nutrient salt conditions
Skeletons of diatoms (Skeletonema costatum), red tide heterodrographis (Heterosigma akashiwo), and heterodrographis azosii (Heteroapaassteini) 3 typical bait algae were selected. Noctilucent algae were co-cultivated with 3 bait algae, respectively, according to the method of example 2. And 3 nutrient salt concentrations, respectively f/2, f/8 and f/32, are set for the co-culture system, and the same amount of natural seawater is added as a control group. The experiment uses a 100mL conical flask, the culture volume of each treatment group is 50mL, and the initial cell densities of noctiluca, isodon japonicus, isodon akabane and Skeletonema costatum are 5 and 1 multiplied by 10 respectively 4 、1×10 4 、4.2×10 5 cells/mL, 3 biological replicates per experimental group were set. The culture temperature is 20 ℃, and the light-dark period is 14h: the time period of the reaction is 10 hours,the illumination intensity is 100+/-10 mu E.m -2 ·s -1 During which bait is not fed. And (5) counting cells of noctilucent algae and bait algae respectively every 2 d.
The results show that as the concentration of nutrient salts in the algae liquid increases, the growth of 3 typical bait algae is faster. At the 6d of the culture period, the cell densities of the Isodon stonecrop, the Isodon akabane and the Skeletonema costatum in the treatment group with the nutrient salt concentration of f/2 were respectively from 1.0X10 4 、1.0×10 4 And 4.2X10 4 The cell/mL was raised to (1.5.+ -. 0.1). Times.10 5 、(3.6±0.1)×10 5 And (5.4.+ -. 0.1). Times.10 5 cells/mL (FIGS. 1 and 4-5). In 3 experimental groups, noctiluca algae grows most rapidly in a coculture system with the heterodera schneideriana, and the noctiluca algae population density of the f/2-Si treatment group is increased to (109+/-3.9) cells/mL in the 6d and is obviously higher than that of other culture systems; and its population density increases with increasing xenobiotic gracilis population density, and more and full food bubbles are observed in its body (fig. 1, fig. 6A). In the co-culture system with the red tide heterotrima, the density of noctiluca species increases with the density of the red tide heterotrima species, and a plurality of food bubbles exist in the body. At the 6d of the culture period, the population density of the f/2-Si treated group noctiluca was increased to (38.6.+ -. 3.2) cells/mL (FIG. 4, FIG. 6C). And when co-cultured with skeletonema costatum, noctiluca gregata grew slowly in each treatment group. Wherein, the population density of noctilucent algae in the f/2 treatment group is reduced at the 4d, the 6d is reduced to (11.6+/-0.8) cells/mL, and the food bubbles are hardly existed in the body (fig. 5 and fig. 6B). Wherein the scale in the A-C diagram in FIG. 6 is 100. Mu.m.

Claims (8)

1. A method for stably proliferating noctilucent algae based on mixed culture is characterized in that Red noctilucent algae (Red Noctiluca scintillans) He s's heterodera avenueHeterocapsa steinii) Co-culturing; the co-cultured system contains nutrient salts including N element, si element, P element, trace metal elements and vitamins.
2. According to claimThe method according to 1, wherein the substance containing N element comprises NaNO 3 The Si-containing substance includes Na 2 SiO 3 •9H 2 O, the substance containing the P element comprises NaH 2 PO 4 •2H 2 O。
3. The method of claim 1, wherein the trace metal element comprises one or more of Fe, cu, zn, co, mn or Mo.
4. The method according to claim 3, wherein the substance containing Fe comprises FeCl 3 •6H 2 O, the substance containing Cu comprises CuSO 4 •5H 2 O, the Zn-containing substance comprises ZnSO 4 •7H 2 O, the substance containing the Co comprises CoCl 2 •6H 2 O, the substances containing Mn include MnCl 2 •4H 2 O, the Mo-containing material including Na 2 MoO 4 •2H 2 O。
5. The method of claim 1, wherein the vitamin comprises vitamin B 12 H or B 1 One or more of them.
6. The method of claim 2, wherein the NaNO 3 The concentration of (2) is 0.551×10 -4 ～8.82×10 -4 mol/L of the Na 2 SiO 3 •9H 2 The concentration of O is 0.663 ×10 -5 ～1.06×10 -4 mol/L, the NaH 2 PO 4 •2H 2 O concentration of 0.226×10 -5 ～3.62×10 -5 mol/L。
7. The method of claim 4, wherein the feci 3 •6H 2 O concentration of 0.731×10 -6 ～1.17×10 -5 mol/L, the CuSO 4 •5H 2 O concentration of 0.246×10 -8 ～3.93×10 -8 mol/L, the ZnSO 4 •7H 2 O concentration of 0.478×10 -8 ～7.65×10 -8 mol/L of the CoCl 2 •6H 2 O concentration of 0.263×10 -8 ～4.2×10 -8 mol/L of MnCl 2 •4H 2 O concentration of 0.569×10 -7 ～9.1×10 -7 mol/L of the Na 2 MoO 4 •2H 2 O concentration of 0.163×10 -8 ～2.6×10 -8 mol/L。
8. The method of claim 5, wherein the vitamin B 12 Is 0.231×10 in concentration -10 ～3.69×10 -10 mol/L, the concentration of vitamin H is 0.128×10 -9 ～2.05×10 -9 mol/L of the vitamin B 1 Is 0.185×10 in concentration -7 ～2.96×10 -7 mol/L。
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