CN109182132A - Produce the cultural method of the Alexandrium mimutum Halim of gonyatoxin - Google Patents
Produce the cultural method of the Alexandrium mimutum Halim of gonyatoxin Download PDFInfo
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Abstract
The invention discloses the cultural method for the Alexandrium mimutum Halim for producing gonyatoxin, Alexandrium mimutum Halim adds ocean rhodotorula extract using conventional cultural method and condition, and in Alexandrium mimutum Halim Initial stage of culture in the medium;Ocean rhodotorula extract the preparation method comprises the following steps: take ocean rhodotorula dry powder, distilled water, (S)-(+) -3- (1- naphthoxy) -1- phenyl-1-propanol and citric acid is added, suspension is made, ultrasound;PH value of solution, proper temperature are adjusted, papain, enzymatic hydrolysis is added;Centrifugation, takes supernatant to be freeze-dried, obtains ocean rhodotorula extract.The utility model has the advantages that incubation is not susceptible to pollute, and Alexandrium mimutum Halim growth rate is fast, it is high to produce malicious content the present invention provides a kind of cultural method of the Alexandrium mimutum Halim of production gonyatoxin being simple and efficient.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to the culture side for the Alexandrium mimutum Halim for producing gonyatoxin
Method.
Technical background
It is found for the first time from nineteen fifty-seven saxitoxin (Saxitoxin STX), shares 57 kinds of paralytic shellfish poisoning (PSP)s
(paralytic shellfish toxins PSTs) homologue is described.
Paralytic shellfish poisoning (PSP) is a kind of alkaloid with neural paralysis effect, and mechanism of poisoning is that toxin combines sodium
Ion channel structure hinders the flowing of sodium ion.There is no antidote after poisoning, unique rescue method is artificial respiration and defeated
Liquid.PSTs militarily has a possibility that applied to chemical and biological weapons because its toxicity is strong, colorless and odorless and has paralysis effect,
Medically then as the precursor substance for exploring Novel anesthetic, and in terms of environment and food inspection, the existing part of foreign countries
PSTs toxin standard items are sold, but price is expensive.
Paralytic shellfish poisoning (PSP) is widely used in medical square mask.One side environmental protection, food security aspect need
The harm of this toxoid is prevented, on the other hand, due to its strong physiological activity, and can be used.It is medicinal in toxin
Aspect is similar to conotoxin, using paralytic shellfish poisoning (PSP) as lead compound, the high biological products of exploitation small toxicity, activity
It is one of the hot spot of current research.
Due to paralysis effect, STX therefore the possibility with exploitation Novel anesthetic.It, can in order to overcome toxicity problem
Liposome reagent is made in STX, STX liposome reagent has the characteristic of slow release, is expected to be applied to clinic.Gonyatoxin
GTX2/3 equally has clinical potential, and it is to compare safely effectively that they, which are used to treatment anal fissure, and it is slow can also to be used to treatment
The headache of sexual tension type.As more and more toxin are found, the application value of paralytic shellfish poisoning (PSP) can be promoted further,
It shows good application prospect.
Paralytic shellfish poisoning (PSP) occurs mainly with microalgae, is probed by producing malicious factor to harmful microalgae, people can be more
The outburst and influence of early warning harmful algal well reduces property loss and health risk;It is high that breeding Toxin producing C can also be explored
Toxiferous algae, prepare mark product, be applied to scientific research and medical test.Therefore, to the growth of harmful algae for producing paralytic shellfish poisoning (PSP)
Corresponding research important in inhibiting is carried out with malicious influence factor is produced.
Alexandrium mimutum Halim (Alexandrium minutum) is subordinate to Protista (Protoctista), Pyrrhophyta
(Dinoflagellata), Alexandria belongs to (Alexandrium), is a kind of Marine Planktonic dinoflagellate for having first piece, cell size
At 13-30 μm, it is widely distributed and with produce paralytic shellfish poisoning (PSP) (paralytic shellfishtoxins PSTs) red tide
It is related.Its gonyatoxin generated in vivo (gonyautoxin) GTX1, GTX2, GTX3, GTX4, is enriched with, most through mollusk
Shellfish, fish and the mankind are passed to by food chain eventually, the serious paralytic shellfish poisoning (PSP) poisoning (paralytic of people can be caused
Shellfish poisoning PSP), a lot of cause because eating the basket whelk containing paralytic shellfish poisoning (PSP) extremely has occurred for the country
The accident died.
However but just there is very big notch for paralytic shellfish poisoning (PSP) at present, since acquisition modes are more single,
The toxin obtained at present to the acquisition modes of paralytic shellfish poisoning (PSP) is far from satisfying needs.It is broader therefore, it is necessary to develop
The efficient method for obtaining toxin.
The prior art such as Authorization Notice No. is the Chinese invention patent of CN 103834567B, discloses a kind of microdisk electrode
Method, the method combine microalgae Immobilized culture and Liquid Culture, and it is enterprising that microalgae is specifically inoculated into solid material
Then row Immobilized culture will be inoculated by the microalgae of Immobilized culture and continue to cultivate in liquid.Use method of the invention
Culture microalgae does not need harsh condition, and energy consumption is lower, is not susceptible to pollute, and can generate highdensity frustule, energy
The industry size metaplasia of the secondary metabolites such as pigment, lipid, hydro carbons and carbohydrate is enough realized by the method that solid-liquid culture combines
It produces.However, this method culture medium complicated composition, micro algae growth rate are slower.
Summary of the invention
The purpose of the present invention is to provide a kind of cultures of the Alexandrium mimutum Halim of production gonyatoxin being simple and efficient
Method, incubation are not susceptible to pollute, and Alexandrium mimutum Halim growth rate is fast, it is high to produce malicious content.
The present invention in background technique aiming at the problem that mentioning, the technical solution taken are as follows:
Produce the cultural method of the Alexandrium mimutum Halim of gonyatoxin, including following content:
Alexandrium mimutum Halim is using conventional cultural method and condition, and in Alexandrium mimutum Halim Initial stage of culture,
Ocean rhodotorula extract is added in culture medium, the amount that ocean rhodotorula extract is added in culture medium is 10-100mg/L.
Preferably, ocean rhodotorula extract the preparation method comprises the following steps: take ocean rhodotorula dry powder, rhodotorula dry powder is added
(S)-(+) -3- (1- the naphthoxy) -1- phenyl -1- third of 2-5 times of quality of distilled water, rhodotorula dry powder quality 0.03-0.08%
The citric acid of pure and mild 0.02-0.05%, is made suspension, the ultrasound 10-40min under power 500-600W;Adjust pH to 5.0-
6.5,45-65 DEG C of temperature, the papain of rhodotorula dry powder quality 0.1-1% is added, digests 6-24h;Centrifugation, takes supernatant
Freeze-drying, obtains ocean rhodotorula extract;(S)-(+) -3- (1- naphthoxy) -1- phenyl-1-propanol and citric acid have coupling
Cooperation is used, and under ultrasonication, can promote ocean rhodotorula cell rupture, so that intracellular beta glucan, manna oligosacchride, work
Property polypeptide and astaxanthin isoreactivity substance dissolution, abundant nutriment is provided for Alexandrium mimutum Halim growth and breeding, together
When active peptides be able to suppress inflammation and resist microorganism infection, be conducive to Alexandrium mimutum Halim Initial stage of culture just builds be good for
The culture medium system of health harmony, to improve culture efficiency.
Preferably, the condition of culture of Alexandrium mimutum Halim are as follows: 15-50 DEG C of temperature, 100-600 μm of ol/ of intensity of illumination
m2/ s, incubation time 2-10 days.
Preferably, culture is carried out in endless totally-enclosed or open environment;Endless totally enclosed environment is such as divulged information
Glass greenhouse etc., open environment such as raceway pond etc.;Using semi-continuous training method, it can investigate and adjust in time and is micro-
The conditional parameter of biological culture is conducive to seek optimal culture environment, improves culture efficiency, and have cultivation cycle short, light
The advantages that utilization rate is high.
Preferably, producing the Alexandrium mimutum Halim culture medium of gonyatoxin, each of following mass concentration is specifically included
Component: NaNO3For 1.1-1.8g/L, K2HPO4·3H2O is 0.02-0.05g/L, MgSO4·7H2O is 0.05-0.08g/L,
ZnSO4For 0.02-0.04g/L, CoCl2For 0.001-0.004g/L, MnCl2For 0.02-0.04g/L, FeCl3·6H2O is
0.002-0.005g/L, Na2EDTA·2H2O is 0.001-0.003g/L, citric acid 0.003-0.006g/L, natural sea-water.
Further preferably, natural sea-water does following processing: adding the lauric acid and 0.006- of 0.02-0.03 ‰ in the seawater
0.01% potassium sorbate, stirs evenly, and filters after standing 24-36h;The special presence of lauric acid and potassium sorbate is able to suppress
Harmful microbe dehydrogenase system in seawater makes protein coagulating and the denaturation of microorganism, to inhibit harmful microbe
Existence and breeding, provide health advantageous environment for the building of culture medium, be capable of the stability of intensified education base composition, thus
The resistance to growth for improving culture medium is conducive to the numerous benefit of expansion for improving Alexandrium mimutum Halim, avoids because pollution etc. is unstable
The waste of cost caused by factor;Meanwhile may advantageously facilitate the growth rate of Alexandrium mimutum Halim, small Asia finally can be improved and go through
The big algae in mountain produces malicious content.
Compared with the prior art, the advantages of the present invention are as follows: 1) present invention in Alexandrium mimutum Halim Initial stage of culture training
It supports and adds ocean rhodotorula extract in base, ocean rhodotorula extract is rich in beta glucan, manna oligosacchride, activity into the cell
Polypeptide and astaxanthin isoreactivity substance, can provide abundant nutriment, simultaneously for Alexandrium mimutum Halim growth and breeding
Active peptides are able to suppress inflammation and resist microorganism infection, are conducive to just build health in Alexandrium mimutum Halim Initial stage of culture
Harmonious culture medium system, to improve culture efficiency;2) it is added in Alexandrium mimutum Halim medium component natural sea-water
Lauric acid and potassium sorbate are able to suppress harmful microbe dehydrogenase system in seawater, make microorganism protein coagulating and
Denaturation provides health advantageous environment, Neng Goujia to inhibit the existence and breeding of harmful microbe for the building of culture medium
The stability of strong culture medium composition is conducive to the expansion for improving Alexandrium mimutum Halim to improve the resistance to growth of culture medium
Numerous benefit, cost wastes caused by avoiding because of unstable factors such as pollutions;Meanwhile it may advantageously facilitate the life of Alexandrium mimutum Halim
Long rate finally can be improved Alexandrium mimutum Halim and produce malicious content.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
Investigate influence of the ocean rhodotorula extractive content to Alexandrium mimutum Halim culture.
Alexandrium mimutum Halim algae solution in logarithmic growth phase is inoculated in the open raceway pond reactor of 20L,
Culture medium includes each component of following mass concentration: NaNO3For 1.6g/L, K2HPO4·3H2O is 0.03g/L, MgSO4·7H2O
For 0.06g/L, ZnSO4For 0.03g/L, CoCl2For 0.002g/L, MnCl2For 0.04g/L, FeCl3·6H2O is 0.004g/L,
Na2EDTA·2H2O is 0.002g/L, citric acid 0.005g/L, natural sea-water;Alexandrium mimutum Halim is initial close after inoculation
Degree is 1 × 106A/mL, the ocean rhodotorula extract for adding different content carry out culture experiment;Experiment is trained at 30 DEG C
It supports, 350 μm of ol/m of intensity of illumination2/ s, for 24 hours, brightness time ratio is that 14 ﹕ 10 (hour) use blood cell after culture 6 days to light dark period
Counting method carries out observation counting, gained to the quantity of the quantity of Alexandrium mimutum Halim and miscellaneous bacteria and pest and disease damage under the microscope
Result such as table 1;
Above-mentioned natural sea-water does following processing: 0.02 ‰ lauric acid and 0.007% potassium sorbate are added in the seawater,
It stirs evenly, standing filters afterwards for 24 hours;It is de- that the special presence of lauric acid and potassium sorbate is able to suppress harmful microbe in seawater
Hydrogen enzyme system, makes protein coagulating and the denaturation of microorganism, is culture medium to inhibit the existence and breeding of harmful microbe
Building provide health advantageous environment, be capable of the stability of intensified education base composition, to improve the resistance to growth of culture medium
Property, be conducive to the numerous benefit of expansion for improving Alexandrium mimutum Halim, cost wastes caused by avoiding because of unstable factors such as pollutions;Together
When, it may advantageously facilitate the growth rate of Alexandrium mimutum Halim, Alexandrium mimutum Halim finally can be improved and produce malicious content;
Ocean rhodotorula extract the preparation method comprises the following steps: take ocean rhodotorula dry powder, be added 4 times of rhodotorula dry powder quality
Distilled water, (S)-(+) -3- (1- the naphthoxy) -1- phenyl-1-propanol of rhodotorula dry powder quality 0.05% and 0.03% lemon
Suspension is made in acid, the ultrasound 30min under power 550W;PH to 6.5 is adjusted, rhodotorula dry powder quality is added in temperature 50 C
0.6% papain digests 20h;Centrifugation, takes supernatant to be freeze-dried, obtains ocean rhodotorula extract;(S)-(+)-3-
There is coupling can speed up ocean rhodotorula under ultrasonication for (1- naphthoxy) -1- phenyl-1-propanol and citric acid
Cell rupture, so that intracellular beta glucan, manna oligosacchride, active peptides and the dissolution of astaxanthin isoreactivity substance, are small
Alexandrium growth and breeding provides abundant nutriment, while active peptides are able to suppress inflammation and resist microorganism sense
Dye is conducive to just build the culture medium system of healthy harmony in Alexandrium mimutum Halim Initial stage of culture, to improve culture efficiency.
Influence of the 1 ocean rhodotorula extractive content of table to Alexandrium mimutum Halim culture
Embodiment 2:
Investigate the influence of temperature, light intensity to Alexandrium mimutum Halim culture.
Alexandrium mimutum Halim algae solution in logarithmic growth phase is inoculated in the open raceway pond reactor of 20L,
Culture medium includes each component of following mass concentration: NaNO3For 1.6g/L, K2HPO4·3H2O is 0.03g/L, MgSO4·7H2O
For 0.06g/L, ZnSO4For 0.03g/L, CoCl2For 0.002g/L, MnCl2For 0.04g/L, FeCl3·6H2O is 0.004g/L,
Na2EDTA·2H2O is 0.002g/L, citric acid 0.005g/L, natural sea-water;Alexandrium mimutum Halim is initial close after inoculation
Degree is 1 × 106A/mL, the ocean rhodotorula extract for adding 80mg/L carry out culture experiment;Cultivate light dark period for 24 hours, brightness
Time ratio be 14 ﹕ 10 (hour), culture 6 days after, with counting method of blood cell under the microscope to the quantity of Alexandrium mimutum Halim with
And the quantity of miscellaneous bacteria and pest and disease damage carries out observation counting, resulting result such as table 2;
Above-mentioned natural sea-water does following processing: 0.02 ‰ lauric acid and 0.007% potassium sorbate are added in the seawater,
It stirs evenly, is filtered after standing 36h;It is de- that the special presence of lauric acid and potassium sorbate is able to suppress harmful microbe in seawater
Hydrogen enzyme system, makes protein coagulating and the denaturation of microorganism, is culture medium to inhibit the existence and breeding of harmful microbe
Building provide health advantageous environment, be capable of the stability of intensified education base composition, to improve the resistance to growth of culture medium
Property, be conducive to the numerous benefit of expansion for improving Alexandrium mimutum Halim, cost wastes caused by avoiding because of unstable factors such as pollutions;Together
When, it may advantageously facilitate the growth rate of Alexandrium mimutum Halim, Alexandrium mimutum Halim finally can be improved and produce malicious content;
Ocean rhodotorula extract the preparation method comprises the following steps: take ocean rhodotorula dry powder, be added 4 times of rhodotorula dry powder quality
Distilled water, (S)-(+) -3- (1- the naphthoxy) -1- phenyl-1-propanol of rhodotorula dry powder quality 0.05% and 0.03% lemon
Suspension is made in acid, the ultrasound 30min under power 550W;PH to 6.5 is adjusted, rhodotorula dry powder quality is added in temperature 50 C
0.6% papain digests 20h;Centrifugation, takes supernatant to be freeze-dried, obtains ocean rhodotorula extract;(S)-(+)-3-
There is coupling can speed up ocean rhodotorula under ultrasonication for (1- naphthoxy) -1- phenyl-1-propanol and citric acid
Cell rupture, so that intracellular beta glucan, manna oligosacchride, active peptides and the dissolution of astaxanthin isoreactivity substance, are small
Alexandrium growth and breeding provides abundant nutriment, while active peptides are able to suppress inflammation and resist microorganism sense
Dye is conducive to just build the culture medium system of healthy harmony in Alexandrium mimutum Halim Initial stage of culture, to improve culture efficiency.
2 temperature of table, influence of the light intensity to Alexandrium mimutum Halim culture
By embodiment 1-2 it is found that the cultural method of the Alexandrium mimutum Halim of gonyatoxin is produced, when ocean rhodotorula mentions
Taking object additive amount is 80mg/L, and cultivation temperature is 30 DEG C, and intensity of illumination is 350 μm of ol/m2When/s, the life of Alexandrium mimutum Halim
Length is most advantageous.
Comparative example 1:
Do not added in ocean rhodotorula extract preparation process (S)-(+) -3- (1- naphthoxy) -1- phenyl-1-propanol and
Citric acid, and the additive amount of ocean rhodotorula extract is 80mg/L, rest part and embodiment 1 are completely the same;Thus condition
Under the Alexandrium mimutum Halim biomass that measures be 5.76 × 107A/mL, miscellaneous bacteria and pest and disease damage cell quantity are 1.43 × 106
A/mL shows that (S)-(+) -3- (1- naphthoxy) -1- phenyl-1-propanol and citric acid have coupling, can promote ocean
Red yeast cell rupture, so that intracellular beta glucan, manna oligosacchride, active peptides and the dissolution of astaxanthin isoreactivity substance,
Abundant nutriment is provided for Alexandrium mimutum Halim growth and breeding, while active peptides are able to suppress inflammation and resist micro- life
Object infection, improves culture efficiency.
Comparative example 2:
It is not added with lauric acid and potassium sorbate in natural sea-water processing, and the additive amount of ocean rhodotorula extract is
80mg/L, rest part and embodiment 1 are completely the same;Thus the Alexandrium mimutum Halim biomass measured under the conditions of be 9.33 ×
107A/mL, miscellaneous bacteria and pest and disease damage cell quantity are 1.25 × 105A/mL, showing lauric acid and potassium sorbate special, there are energy
Enough inhibit the existence and breeding of harmful microbe, provides health advantageous environment for the building of culture medium.
Routine operation in operation of the present invention step is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention,
Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.
Claims (8)
1. producing the cultural method of the Alexandrium mimutum Halim of gonyatoxin, it is characterised in that: in Alexandrium mimutum Halim culture
Initial stage adds ocean rhodotorula extract in the medium.
2. the cultural method of the Alexandrium mimutum Halim according to claim 1 for producing gonyatoxin, it is characterised in that: institute
Stating and adding the amount of ocean rhodotorula extract in culture medium is 10-100mg/L.
3. the cultural method of the Alexandrium mimutum Halim according to claim 1 for producing gonyatoxin, it is characterised in that: institute
State ocean rhodotorula extract the preparation method comprises the following steps: takes ocean rhodotorula dry powder, and distilled water, (S)-(+) -3- (1- naphthalene oxygen is added
Base) -1- phenyl-1-propanol and citric acid, suspension is made, ultrasound;PH to 5.0-6.5 is adjusted, 45-65 DEG C of temperature, is added red
The papain of yeast dry powder quality 0.1-1% digests 6-24h;Centrifugation, takes supernatant to be freeze-dried, obtains ocean rhodotorula
Extract.
4. the cultural method of the Alexandrium mimutum Halim according to claim 3 for producing gonyatoxin, it is characterised in that: institute
State 2-5 times that distilled water additional amount is rhodotorula dry powder quality;(S)-(+) -3- (1- naphthoxy) -1- phenyl-1-propanol sum
Citric acid adding amount is respectively the 0.03-0.08% and 0.02-0.05% of rhodotorula dry powder quality.
5. the cultural method of the Alexandrium mimutum Halim according to claim 1 for producing gonyatoxin, it is characterised in that: institute
State the condition of culture of Alexandrium mimutum Halim are as follows: 15-50 DEG C of temperature, 100-600 μm of ol/m of intensity of illumination2/ s, incubation time 2-
10 days.
6. the cultural method of the Alexandrium mimutum Halim according to claim 1 for producing gonyatoxin, it is characterised in that: institute
Culture is stated to carry out in endless totally-enclosed or open environment.
7. the cultural method of the Alexandrium mimutum Halim according to claim 1 for producing gonyatoxin, it is characterised in that: institute
State each component that culture medium includes following mass concentration: NaNO3For 1.1-1.8g/L, K2HPO4·3H2O is 0.02-0.05g/L,
MgSO4·7H2O is 0.05-0.08g/L, ZnSO4For 0.02-0.04g/L, CoCl2For 0.001-0.004g/L, MnCl2For
0.02-0.04g/L, FeCl3·6H2O is 0.002-0.005g/L, Na2EDTA·2H2O is 0.001-0.003g/L, citric acid
For 0.003-0.006g/L, natural sea-water.
8. the cultural method of the Alexandrium mimutum Halim according to claim 7 for producing gonyatoxin, it is characterised in that: institute
It states natural sea-water and does following processing: adding the lauric acid of 0.02-0.03 ‰ and the potassium sorbate of 0.006-0.01% in the seawater,
It stirs evenly, is filtered after standing 24-36h.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN116396863A (en) * | 2023-04-13 | 2023-07-07 | 自然资源部第一海洋研究所 | Alexander algae cultivation method |
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