CN107593416A - A kind of cryopreservation method of spirulina algae kind - Google Patents
A kind of cryopreservation method of spirulina algae kind Download PDFInfo
- Publication number
- CN107593416A CN107593416A CN201710689237.9A CN201710689237A CN107593416A CN 107593416 A CN107593416 A CN 107593416A CN 201710689237 A CN201710689237 A CN 201710689237A CN 107593416 A CN107593416 A CN 107593416A
- Authority
- CN
- China
- Prior art keywords
- spirulina
- temperature
- under
- illumination
- conditions
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of cryopreservation method of spirulina algae kind, and spirulina algae kind is preserved by way of the nutrient solution with spirulina spore is carried out into stored frozen.After the present invention by the induction of spirulina algae kind by being spirulina spore, cryopreservation is carried out to spirulina spore, it is necessary to during spirulina algae kind, then spirulina spore is thawed and reverts to spirulina algae kind, the survival rate of spirulina algae kind is more than 90%；The spirulina algae kind holding time is long, it is time saving, laborsaving, pollution-free, without genetic drift, while reduce preservation cost.
Description
Technical field：
The present invention relates to a kind of spirulina store method, more particularly to a kind of cryopreservation side of spirulina algae kind
Method.
Background technology：
Spirulina is prokaryotes, by the filamentous individual of a row cellularity, there is loose or tight spiral (see Fig. 1),
So claim spirulina.
Spirulina because have various trace elements, multivitamin, a variety of oligosaccharides and a variety of unrighted acids, and
Praised highly by health products business, worldwide produced；Spirulina is a kind of preferably genetic engineering experiment material simultaneously,
Because spirulina is good compared to the security of Escherichia coli, virus etc., is a kind of healthy prokaryotes.Need to preserve in production pure
Positive algae kind, the research of spirulina is with greater need for pure algae kind pollution-free, without genetic drift.
At present, it is in the field of business both at home and abroad to be all known as：Spirulina is without resting spore (or prothenchyma (of wood)), for the reproduction side of fracture
Formula, in the preservation practice of spirulina, what is used both at home and abroad is all traditional squamous subculture method, and the preservation of conventional squamous subculture
It cannot be guaranteed that it is pollution-free, without genetic drift, during the traditional squamous subculture of spirulina preserves, genetic shift, character be present
The problems such as loss, but also it is time-consuming, laborious, take artificial, and the research on spirulina cryopreservation is rarely reported.
The content of the invention：
In order to solve the problems such as during the traditional squamous subculture of spirulina preserves, genetic shift occurring, character is lost, this
Invention provides a kind of pollution-free, without genetic drift, time saving, laborsaving, inexpensive spirulina cryopreservation method.
The purpose of the present invention is implemented by following technical scheme：A kind of cryopreservation method of spirulina algae kind, pass through by
Nutrient solution with spirulina spore carries out the mode of stored frozen to preserve spirulina algae kind.
Spirulina spore carries out concretely comprising the following steps for stored frozen：The nutrient solution with spirulina spore is carried out just first
Step frost, preliminary freezing temperature are -8~-20 DEG C, and the preliminary frost time is 24~48h, then at a temperature of not higher than -80 DEG C
Freeze to random time.
Preferably, the preliminary freezing temperature is preferably -8~-12 DEG C.
Preferably, add protective agent in the nutrient solution with spirulina spore, the protective agent be carbohydrate protective agent or
Inorganic matter protective agent.
Specifically, the carbohydrate protective agent is any one or more than one in monosaccharide and disaccharide or trisaccharide arbitrarily to compare
The mixing of example；Mass percent concentration of the carbohydrate protective agent in the nutrient solution is 5~10%.
Specifically, the inorganic matter protective agent is glycerine or dimethyl sulfoxide (DMSO)；The inorganic matter protective agent is in the culture
Mass percent concentration in liquid is 5~15%.
In the northern area of China, spirulina algae kind can be changed into spirulina spore in its natural state.Applicant also sends out
The spirulina being now distributed in Ordos, inner Mongolia cultivation greenhouse is survived the winter in the form of spore (see Fig. 2), and by dividing
The comparison of the biological 16-23rDNA in Fig. 1 and Fig. 2 is analysed, 2 be a kind of biology, in other words：Fig. 2 biology is Fig. 1 biologies
Spore state.
Or spirulina algae kind can be changed into spirulina spore using artificial induction's mode, specific method is：
A, the nutrient solution with spirulina algae kind is obtained, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature 14
Under the conditions of~18 DEG C, 2h is induced；It is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 18~24 DEG C, induction
6h, it is 120~129 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 14~18 DEG C, 2h is induced, is 0 in intensity of illumination
~3 μm of ol.m-2.s-1, under the conditions of temperature is 12~14 DEG C, induce 14h；Then the above-mentioned inductive condition in repeat step A lures again
Lead 24h；
B, the nutrient solution of step A inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature be 10~
Under the conditions of 14 DEG C, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 14~16 DEG C, induction
6h, it is 120~129 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 10~14 DEG C, 2h is induced, is 0 in intensity of illumination
~3 μm of ol.m-2.s-1, under the conditions of temperature is 8~10 DEG C, induce 14h；Then the above-mentioned inductive condition in repeat step B lures again
Lead 24h；
C, the nutrient solution of step B inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 4~6
Under the conditions of DEG C, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 6~8 DEG C, 6h is induced,
Intensity of illumination is 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 4~6 DEG C, 2h is induced, is 0~3 μ in intensity of illumination
mol.m-2.s-1, under the conditions of temperature is 0~3 DEG C, induce 14h；Then the above-mentioned inductive condition in repeat step C induces 24h again；
D, the nutrient solution of step C inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 3~4
Under the conditions of DEG C, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 4 DEG C, 6h is induced, in illumination
Intensity is 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 3~4 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination- 2.s-1, temperature is 0~2 DEG C, induces 14h；Then the above-mentioned inductive condition in repeat step D induces 24h again, now, such as spiral
Algae algae kind is changed into spirulina spore, then terminates to induce；As spirulina algae kind is not changed into spirulina spore, then continue step E
Induction；
E, step D inductions will be completed and be not changed into the nutrient solution of spirulina spore, be 120~129 μ in intensity of illumination
mol.m-2.s-1, under the conditions of temperature is 1~2 DEG C, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, temperature is 2 DEG C
Under the conditions of, 6h is induced, is 120~129 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 1~2 DEG C, 2h is induced, in light
It is 0~3 μm of ol.m according to intensity-2.s-1, under the conditions of temperature is 0 DEG C, induce 14h；Then the above-mentioned inductive condition in repeat step E
Circulation-induced, until spirulina algae kind is changed into spirulina spore, terminate induction.
The cryopreservation method of described spirulina, also include defrosting step, specifically, first, the spiral shell that will be frozen
Algae spore is revolved, thaw 92h at a temperature of going to -12 DEG C, then, is more than 0 DEG C in minimum temperature, maximum temperature is not more than 2 DEG C of conditions
After lower defrosting is melted completely to ice, finally, 8 DEG C are not higher than in maximum temperature, minimum temperature is cultivated under the conditions of being not less than 0 DEG C, directly
Spirulina algae kind is reverted to spirulina spore.
Advantages of the present invention：The present invention is by the way that the induction of spirulina algae kind after spirulina spore, is entered to spirulina spore
Row cryopreservation is, it is necessary to during spirulina algae kind, then spirulina spore is thawed and reverts to spirulina algae kind, spirulina algae kind
Survival rate is more than 90%；The spirulina algae kind holding time is long, it is time saving, laborsaving, pollution-free, without genetic drift, reduce simultaneously
Preserve cost.
Brief description of the drawings：
Fig. 1 is the thread individual state figure of spirulina algae kind.
Fig. 2 is that spirulina spore amplifies the picture under 100 times in microscope.
Embodiment：
Embodiment 1：A kind of cryopreservation method of spirulina algae kind, it comprises the following steps：
(1) it is spirulina spore by the induction of spirulina algae kind, specific method is：Obtained at the northern area of China, autumn
Nutrient solution with spirulina algae kind, and add monose in nutrient solution and cook protective agent, quality percentage of the monose in nutrient solution
Specific concentration is 5%, is then cultivated in its natural state, to winter, spirulina algae kind natural temperature and illumination condition in the winter time
Induction under, be changed into spirulina spore.
(2) the spirulina spore stored frozen for obtaining step (1), is concretely comprised the following steps：Spirulina spore is carried out first
Preliminary frost, preliminary freezing temperature are -8 DEG C, and the preliminary frost time is 48h, is then frozen at a temperature of -80 DEG C to 6 months.
(3) the spirulina spore by step (2) frost thaws, and reverts to spirulina algae kind, concretely comprises the following steps：Will be -80
The spirulina spore frozen at a temperature of DEG C, thaw 92h at a temperature of going to -12 DEG C, then, is more than 0 DEG C in minimum temperature, the highest temperature
Degree thaws under the conditions of being not more than 2 DEG C to be melted completely to ice, finally, is not higher than 8 DEG C in maximum temperature, minimum temperature is not less than 0 DEG C
Under the conditions of cultivate, until spirulina spore reverts to spirulina algae kind.
Detection contrast is carried out to the amount of survival of the spirulina algae kind before freezing and after recovery, spirulina algae kind is chilled
Afterwards, its survival rate is 98%.
Embodiment 2：A kind of cryopreservation method of spirulina algae kind, it comprises the following steps：
(1) it is spirulina spore by the induction of spirulina algae kind, specific method is：Obtained at the northern area of China, autumn
Nutrient solution with spirulina algae kind, addition trisaccharide cooks protective agent, mass percent of the trisaccharide in nutrient solution in nutrient solution
Concentration is 8%, is then cultivated in its natural state, to winter, spirulina algae kind natural temperature and illumination condition in the winter time
Under induction, it is changed into spirulina spore.
(2) the spirulina spore stored frozen for obtaining step (1), is concretely comprised the following steps：Spirulina spore is carried out first
Preliminary frost, preliminary freezing temperature are -12 DEG C, and the preliminary frost time is 36h, is then frozen at a temperature of -100 DEG C to 12
Month.
(3) the spirulina spore by step (2) frost thaws, and reverts to spirulina algae kind, concretely comprises the following steps：Will be -80
The spirulina spore frozen at a temperature of DEG C, thaw 92h at a temperature of going to -12 DEG C, then, is more than 0 DEG C in minimum temperature, the highest temperature
Degree thaws under the conditions of being not more than 2 DEG C to be melted completely to ice, finally, is not higher than 8 DEG C in maximum temperature, minimum temperature is not less than 0 DEG C
Under the conditions of cultivate, until spirulina spore reverts to spirulina algae kind.
Detection contrast is carried out to the amount of survival of the spirulina algae kind before freezing and after recovery, spirulina algae kind is chilled
Afterwards, its survival rate is 100%.
Embodiment 3：A kind of cryopreservation method of spirulina algae kind, it comprises the following steps：
(1) it is spirulina spore by the induction of spirulina algae kind, specific method is：Obtained at the northern area of China, autumn
Nutrient solution with spirulina algae kind, addition dimethyl sulfoxide (DMSO) cooks protective agent in nutrient solution, and dimethyl sulfoxide (DMSO) is in nutrient solution
Mass percent concentration be 15%；Then cultivate in its natural state, to winter, spirulina algae kind natural temperature in the winter time
Under the induction of illumination condition, it is changed into spirulina spore.
(2) the spirulina spore stored frozen for obtaining step (1), is concretely comprised the following steps：Spirulina spore is carried out first
Preliminary frost, preliminary freezing temperature are -20 DEG C, and the preliminary frost time is 24h, is then frozen in liquid nitrogen to 18 months.
(3) the spirulina spore by step (2) frost thaws, and reverts to spirulina algae kind, concretely comprises the following steps：Will be -80
The spirulina spore frozen at a temperature of DEG C, thaw 92h at a temperature of going to -12 DEG C, then, is more than 0 DEG C in minimum temperature, the highest temperature
Degree thaws under the conditions of being not more than 2 DEG C to be melted completely to ice, finally, is not higher than 8 DEG C in maximum temperature, minimum temperature is not less than 0 DEG C
Under the conditions of cultivate, until spirulina spore reverts to spirulina algae kind.
Detection contrast is carried out to the amount of survival of the spirulina algae kind before freezing and after recovery, spirulina algae kind is chilled
Afterwards, its survival rate is 99%.
Embodiment 4：A kind of cryopreservation method of spirulina algae kind, it comprises the following steps：
(1) it is spirulina spore by the induction of spirulina algae kind, specific method is：
A, the nutrient solution with spirulina algae kind is obtained, disaccharide is added in nutrient solution and is being cultivated as protective agent, disaccharide
Mass percent concentration in liquid is 5%；Then, it is 120~129 μm of ol.m in intensity of illumination-2.s-1, equivalent to 2 40w's
The intensity of fluorescent tube irradiation, under the conditions of temperature is 14 DEG C, induce 2h；It is 230~239 μm of ol.m in intensity of illumination-2.s-1, equivalent to
The exposure intensity of 4 40w fluorescent tube, under the conditions of temperature is 18 DEG C, 6h is induced, is 120~129 μm of ol.m in intensity of illumination-2.s-1, equivalent to 2 40w fluorescent tube exposure intensity, under the conditions of temperature is 14 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, the intensity of light under light dark state is equivalently employed without, under the conditions of temperature is 12 DEG C, induces 14h；Then repeat step A
In above-mentioned inductive condition induce 24h again；
B, the nutrient solution of step A inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 10 DEG C
Under the conditions of, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 14 DEG C, 6h is induced, in illumination
Intensity is 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 10 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1,
Under the conditions of temperature is 8 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step B induces 24h again；
C, the nutrient solution of step B inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 4 DEG C of bars
Under part, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 6 DEG C, 6h is induced, in intensity of illumination
For 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 4 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, temperature
Under the conditions of 0 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step C induces 24h again；
D, the nutrient solution of step C inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 3 DEG C of bars
Under part, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 4 DEG C, 6h is induced, in intensity of illumination
For 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 3 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, temperature
For 0 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step D induces 24h again, and now, spirulina algae kind is changed into spiral shell
Algae spore is revolved, terminates induction；
(2) the spirulina spore stored frozen for obtaining step (1), is concretely comprised the following steps：Spirulina spore is carried out first
Preliminary frost, preliminary freezing temperature are -8 DEG C, and the preliminary frost time is 48h, is then frozen at a temperature of -80 DEG C to 6 months.
(3) the spirulina spore by step (2) frost thaws, and reverts to spirulina algae kind, concretely comprises the following steps：Will be -80
The spirulina spore frozen at a temperature of DEG C, thaw 92h at a temperature of going to -12 DEG C, then, is more than 0 DEG C in minimum temperature, the highest temperature
Degree thaws under the conditions of being not more than 2 DEG C to be melted completely to ice, finally, is not higher than 8 DEG C in maximum temperature, minimum temperature is not less than 0 DEG C
Under the conditions of cultivate, until spirulina spore reverts to spirulina algae kind.
Detection contrast is carried out to the amount of survival of the spirulina algae kind before freezing and after recovery, spirulina algae kind is chilled
Afterwards, its survival rate is 97%.
Embodiment 5：A kind of cryopreservation method of spirulina algae kind, it comprises the following steps：
(1) it is spirulina spore by the induction of spirulina algae kind, specific method is：
A, the nutrient solution with spirulina algae kind is obtained, glycerine is added in nutrient solution and is being cultivated as protective agent, glycerine
Mass percent concentration in liquid is 10%；Then, it is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 16 DEG C of bars
Under part, 2h is induced；It is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 20 DEG C, 6h is induced, it is strong in illumination
Spend for 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 16 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, temperature
Spend under the conditions of 13 DEG C, to induce 14h；Then the above-mentioned inductive condition in repeat step A induces 24h again；
B, the nutrient solution of step A inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 12 DEG C
Under the conditions of, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 15 DEG C, 6h is induced, in illumination
Intensity is 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 12 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1,
Under the conditions of temperature is 9 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step B induces 24h again；
C, the nutrient solution of step B inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 5 DEG C of bars
Under part, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 7 DEG C, 6h is induced, in intensity of illumination
For 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 5 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, temperature
Under the conditions of 2 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step C induces 24h again；
D, the nutrient solution of step C inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 3 DEG C of bars
Under part, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 4 DEG C, 6h is induced, in intensity of illumination
For 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 3 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, temperature
For 1 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step D induces 24h again；
E, step D inductions will be completed and be not changed into the nutrient solution of spirulina spore, be 120~129 μ in intensity of illumination
mol.m-2.s-1, under the conditions of temperature is 1 DEG C, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, temperature is 2 DEG C of bars
Under part, 6h is induced, is 120~129 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 1 DEG C, 2h is induced, in intensity of illumination
For 0~3 μm of ol.m-2.s-1, under the conditions of temperature is 0 DEG C, induce 14h；Then the above-mentioned inductive condition circulation in repeat step E lures
Lead, until spirulina algae kind is changed into spirulina spore, terminate induction；
(2) the spirulina spore stored frozen for obtaining step (1), is concretely comprised the following steps：Spirulina spore is carried out first
Preliminary frost, preliminary freezing temperature are -12 DEG C, and the preliminary frost time is 36h, is then frozen at a temperature of -80 DEG C to 12 months.
(3) the spirulina spore by step (2) frost thaws, and reverts to spirulina algae kind, concretely comprises the following steps：Will be -80
The spirulina spore frozen at a temperature of DEG C, thaw 92h at a temperature of going to -12 DEG C, then, is more than 0 DEG C in minimum temperature, the highest temperature
Degree thaws under the conditions of being not more than 2 DEG C to be melted completely to ice, finally, is not higher than 8 DEG C in maximum temperature, minimum temperature is not less than 0 DEG C
Under the conditions of cultivate, until spirulina spore reverts to spirulina algae kind.
Detection contrast is carried out to the amount of survival of the spirulina algae kind before freezing and after recovery, spirulina algae kind is chilled
Afterwards, its survival rate is 90%.
Embodiment 6：A kind of cryopreservation method of spirulina algae kind, it comprises the following steps：
(1) it is spirulina spore by the induction of spirulina algae kind, specific method is：
A, the nutrient solution with spirulina algae kind is obtained, it is preferred that trisaccharide is added in nutrient solution as protective agent, trisaccharide
Mass percent concentration in nutrient solution is 8%, is 120~129 μm of ol.m in intensity of illumination then-2.s-1, temperature 18
Under the conditions of DEG C, 2h is induced；It is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 24 DEG C, 6h is induced, in light
It is 120~129 μm of ol.m according to intensity-2.s-1, under the conditions of temperature is 18 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination- 2.s-1, under the conditions of temperature is 14 DEG C, induce 14h；Then the above-mentioned inductive condition in repeat step A induces 24h again；
B, the nutrient solution of step A inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 14 DEG C
Under the conditions of, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 16 DEG C, 6h is induced, in illumination
Intensity is 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 14 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1,
Under the conditions of temperature is 10 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step B induces 24h again；
C, the nutrient solution of step B inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 6 DEG C of bars
Under part, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 8 DEG C, 6h is induced, in intensity of illumination
For 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 6 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, temperature
Under the conditions of 3 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step C induces 24h again；
D, the nutrient solution of step C inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 3 DEG C of bars
Under part, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 4 DEG C, 6h is induced, in intensity of illumination
For 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 3 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, temperature
For 2 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step D induces 24h again；
E, step D inductions will be completed and be not changed into the nutrient solution of spirulina spore, be 120~129 μ in intensity of illumination
mol.m-2.s-1, under the conditions of temperature is 1 DEG C, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, temperature is 2 DEG C of bars
Under part, 6h is induced, is 120~129 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 1 DEG C, 2h is induced, in intensity of illumination
For 0~3 μm of ol.m-2.s-1, under the conditions of temperature is 0 DEG C, induce 14h；Then the above-mentioned inductive condition circulation in repeat step E lures
Lead, until spirulina algae kind is changed into spirulina spore, terminate induction；
(2) the spirulina spore stored frozen for obtaining step (1), is concretely comprised the following steps：Spirulina spore is carried out first
Preliminary frost, preliminary freezing temperature are -20 DEG C, and the preliminary frost time is 24h, is then frozen in liquid nitrogen to 18 months.
(3) the spirulina spore by step (2) frost thaws, and reverts to spirulina algae kind, concretely comprises the following steps：Will be -80
The spirulina spore frozen at a temperature of DEG C, thaw 92h at a temperature of going to -12 DEG C, then, is more than 0 DEG C in minimum temperature, the highest temperature
Degree thaws under the conditions of being not more than 2 DEG C to be melted completely to ice, finally, is not higher than 8 DEG C in maximum temperature, minimum temperature is not less than 0 DEG C
Under the conditions of cultivate, until spirulina spore reverts to spirulina algae kind.
Detection contrast is carried out to the amount of survival of the spirulina algae kind before freezing and after recovery, spirulina algae kind is chilled
Afterwards, its survival rate is 100%.
The cooling time of spirulina spore is minimum to the survival rate of spirulina algae kind it can be seen from embodiment 1-6,
As can be seen here, the freezing duration of spirulina spore can determine according to being actually needed.
Embodiment 7：Blunt top spirulina (Spirulina platensis) algae kind such as Fig. 1 and the spirulina algae kind are taken respectively
The spirulina spore such as Fig. 2 for inducing to obtain using the inventive method, carry out rDNA (16-23S) detections, PCR amplification spirulinas DNA
Primer be：
F1：GGAGGCTGTGAAGAATTCCTTGTGG
R1：GAA CTG TCT CAC GAC GTT CTG AAC C have respectively.6 repetitions, sequencing company are the auspicious sound in Shanghai
Company.
The base sequence of blunt top spirulina (Spirulina platensis) algae kind is as follows：
GGGAGGCTGTGAAGAATTCCTTGTGGGGGATTCTGAGCCATCGGTGAGATACCACTCTGGCGAAGCTAG
AATTCTAACCTTTTCCCGTTATCCGGGAAAGAGACAGTTTCAGGGGGGCAGTTTGACTGGGGCGGTCGCCTCCTAAA
TGGTAACGGAGGCGCGCAAGGGTTCCCTCAGGCTGGTTGGAAATCAGCCGTAGAGTGCAAAGGCATAAGGGAGCTTG
ACTGCAAGACCTACAAGTCGAGCAGGGACGAAAGTCGGCCTTAGTGATCCGACGGTGCTGCGTGGAAAGGCCGTCGC
TCAACGGATAAAAGTTACTCTAGGGATAACAGGCTGATCTCCCCCAAGAGTTCACATCGACGGGGAGGTTTGGCACC
TCGATGTCGGCTCATCGCAACCTGGGGCGGTAGTACGTCCCAAGGGTTGGGCTGTTCGCCCATTAAAGCGGTACGTG
AGCTGGGTTCAGAACGTCGTGAGACAGTTC
The base sequence of blunt top spirulina (Spirulina platensis) spore is as follows：
GGGAGGCTGTGAAGAATTCCTTGTGGGGGATTCTGAGCCATCGGTGAGATACCACTCTGGCGAAGCTAG
AATTCTAACCTTTTCCCGTTATCCGGGAAAGAGACAGTTTCAGGGGGGCAGTTTGACTGGGGCGGTCGCCTCCTAAA
TGGTAACGGAGGCGCGCAAGGGTTCCCTCAGGCTGGTTGGAAATCAGCCGTAGAGTGCAAAGGCATAAGGGAGCTTG
ACTGCAAGACCTACAAGTCGAGCAGGGACGAAAGTCGGCCTTAGTGATCCGACGGTGCTGCGTGGAAAGGCCGTCGC
TCAACGGATAAAAGTTACTCTAGGGATAACAGGCTGATCTCCCCCAAGAGTTCACATCGACGGGGAGGTTTGGCACC
TCGATGTCGGCTCATCGCAACCTGGGGCGGTAGTACGTCCCAAGGGTTGGGCTGTTCGCCCATTAAAGCGGTACGTG
AGCTGGGTTCAGAACGTCGTGAGACAGTTC
Two sections of base sequences contrast visible, blunt top spirulina (Spirulina platensis) algae kind and blunt top spirulina
The base sequence of (Spirulina platensis) spore is identical, it was demonstrated that spirulina algae kind is implicitly present in spore state.
Claims (9)
1. a kind of cryopreservation method of spirulina algae kind, it is characterised in that by by the nutrient solution with spirulina spore
The mode for carrying out stored frozen preserves spirulina algae kind.
A kind of 2. cryopreservation method of spirulina algae kind according to claim 1, it is characterised in that spirulina spore
Carry out concretely comprising the following steps for stored frozen：The nutrient solution with spirulina spore is tentatively freezed first, preliminary frost temperature
Spend for -8~-20 DEG C, the preliminary frost time is 24~48h, is then frozen at a temperature of not higher than -80 DEG C to random time.
3. the cryopreservation method of spirulina according to claim 2, it is characterised in that the preliminary freezing temperature is excellent
Elect -8~-12 DEG C as.
4. the cryopreservation method of a kind of spirulina algae kind according to claim 3, it is characterised in that with spiral
Protective agent is added in the nutrient solution of algae spore, the protective agent is carbohydrate protective agent or inorganic matter protective agent.
5. the cryopreservation method of a kind of spirulina algae kind according to claim 4, it is characterised in that the carbohydrate is protected
It is any one or more than one in monosaccharide and disaccharide or trisaccharide with the mixing of arbitrary proportion to protect agent；The carbohydrate protective agent exists
Mass percent concentration in the nutrient solution is 5~10%.
A kind of 6. cryopreservation method of spirulina algae kind according to claim 4, it is characterised in that the inorganic matter
Protective agent is glycerine or dimethyl sulfoxide (DMSO)；Mass percent concentration of the inorganic matter protective agent in the nutrient solution be 5~
15%.
7. the cryopreservation method of the spirulina according to claim 1-6 any one, it is characterised in that in China north
Side area, spirulina algae kind can be changed into spirulina spore in its natural state.
8. the cryopreservation method of the spirulina according to claim 1-6 any one, it is characterised in that spirulina algae
Kind can be changed into spirulina spore using artificial induction's mode, and specific method is：
A, the nutrient solution with spirulina algae kind is obtained, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 14~18
Under the conditions of DEG C, 2h is induced；It is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 18~24 DEG C, 6h is induced,
It is 120~129 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 14~18 DEG C, 2h is induced, is 0~3 μ in intensity of illumination
mol.m-2.s-1, under the conditions of temperature is 12~14 DEG C, induce 14h；Then the above-mentioned inductive condition in repeat step A induces again
24h；
B, the nutrient solution of step A inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 10~14 DEG C of bars
Under part, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 14~16 DEG C, 6h is induced, in light
It is 120~129 μm of ol.m according to intensity-2.s-1, under the conditions of temperature is 10~14 DEG C, 2h is induced, is 0~3 μ in intensity of illumination
mol.m-2.s-1, under the conditions of temperature is 8~10 DEG C, induce 14h；Then the above-mentioned inductive condition in repeat step B induces again
24h；
C, the nutrient solution of step B inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 4~6 DEG C of conditions
Under, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 6~8 DEG C, 6h is induced, it is strong in illumination
Spend for 120~129 μm of ol.m-2.s-1, under the conditions of temperature is 4~6 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1,
Under the conditions of temperature is 0~3 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step C induces 24h again；
D, the nutrient solution of step C inductions will be completed, is 120~129 μm of ol.m in intensity of illumination-2.s-1, temperature is 3~4 DEG C of conditions
Under, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 4 DEG C, 6h is induced, is in intensity of illumination
120~129 μm of ol.m-2.s-1, under the conditions of temperature is 3~4 DEG C, 2h is induced, is 0~3 μm of ol.m in intensity of illumination-2.s-1, temperature
For 0~2 DEG C, 14h is induced；Then the above-mentioned inductive condition in repeat step D induces 24h again, now, as spirulina algae kind changes
For spirulina spore, then terminate to induce；As spirulina algae kind is not changed into spirulina spore, then continue step E induction；
E, step D inductions will be completed and be not changed into the nutrient solution of spirulina spore, be 120~129 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 1~2 DEG C, 2h is induced, is 230~239 μm of ol.m in intensity of illumination-2.s-1, temperature is 2 DEG C of conditions
Under, 6h is induced, is 120~129 μm of ol.m in intensity of illumination-2.s-1, under the conditions of temperature is 1~2 DEG C, 2h is induced, it is strong in illumination
Spend for 0~3 μm of ol.m-2.s-1, under the conditions of temperature is 0 DEG C, induce 14h；Then the above-mentioned inductive condition circulation in repeat step E
Induction, until spirulina algae kind is changed into spirulina spore, terminate induction.
9. according to the cryopreservation method of any described spirulinas of claim 1-6, it is characterised in that it also includes solution
Freeze step, specifically, first, the spirulina spore that will be frozen, thaw 92h at a temperature of going to -12 DEG C, then, in minimum temperature
More than 0 DEG C, maximum temperature is thawed under the conditions of being not more than 2 DEG C melts completely to ice, finally, minimum in maximum temperature not higher than 8 DEG C
Temperature is cultivated under the conditions of being not less than 0 DEG C, until spirulina spore reverts to spirulina algae kind.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710689237.9A CN107593416B (en) | 2017-08-10 | 2017-08-10 | Ultralow-temperature preservation method for spirulina seeds |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710689237.9A CN107593416B (en) | 2017-08-10 | 2017-08-10 | Ultralow-temperature preservation method for spirulina seeds |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107593416A true CN107593416A (en) | 2018-01-19 |
| CN107593416B CN107593416B (en) | 2020-04-10 |
Family
ID=61064766
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710689237.9A Active CN107593416B (en) | 2017-08-10 | 2017-08-10 | Ultralow-temperature preservation method for spirulina seeds |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107593416B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105263320A (en) * | 2013-06-12 | 2016-01-20 | 那慕尔大学 | Freeze preservation of living cells |
| JP2016067235A (en) * | 2014-09-27 | 2016-05-09 | 株式会社ちとせ研究所 | Freezing processing method of algae |
| CN105779292A (en) * | 2016-03-23 | 2016-07-20 | 国家开发投资公司 | Ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment |
-
2017
- 2017-08-10 CN CN201710689237.9A patent/CN107593416B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105263320A (en) * | 2013-06-12 | 2016-01-20 | 那慕尔大学 | Freeze preservation of living cells |
| JP2016067235A (en) * | 2014-09-27 | 2016-05-09 | 株式会社ちとせ研究所 | Freezing processing method of algae |
| CN105779292A (en) * | 2016-03-23 | 2016-07-20 | 国家开发投资公司 | Ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment |
Non-Patent Citations (3)
| Title |
|---|
| 中国美容美体网: "螺旋藻的繁殖方式", 《中国美容美体网》 * |
| 李迷，王素英，董世瑞: "螺旋藻的超低温保存法", 《微生物学通报》 * |
| 郑凌凌等: "藻类种质超低温保藏技术及其应用", 《生物技术》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107593416B (en) | 2020-04-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chancellor et al. | Green onions: potential mechanism for hepatitis A contamination | |
| CN108251361A (en) | A kind of method that umbilical cord tissue block froze, and recovered and prepared mescenchymal stem cell | |
| CN103245647B (en) | Citrus fruit fly odorant binding protein-based attractant screening method | |
| US9382576B2 (en) | Method for isolating and purifying RNA from biological materials | |
| Wang et al. | Cryopreservation of brown algae gametophytes of Undaria pinnatifida by encapsulation–vitrification | |
| CN106942200A (en) | One kind freezes protection liquid and its application | |
| CN108669070A (en) | A kind of cryopreservative solution and its application method of plant tissue and cell | |
| Al-Baba et al. | Cryopreservation and Genetic Stability Assessment of Threatened Medicinal Plant (Ziziphora tenuior L.) Grown Wild in Jordan. | |
| CN107593416A (en) | A kind of cryopreservation method of spirulina algae kind | |
| Fu et al. | Effects of stubble mulch and tillage managements on soil physical properties and water use efficiency of summer maize | |
| Kuwano et al. | Cryopreservation of the conchocelis of the marine alga Porphyra yezoensis Ueda (Rhodophyta) in liquid nitrogen | |
| CN102696580A (en) | Ultralow-temperature storage and regenerative-culture method for embedding and drying of isolated stem tips of test-tube horseradish plantlets | |
| CN111713406A (en) | Embedding vitrification ultra-low temperature detoxification method for Huaiyoshan high-mountain potatoes | |
| CN107058110B (en) | Preservation and recovery method of haematococcus pluvialis cells | |
| CN104126570A (en) | Method for ultralow temperature preservation of Chinese angelica cell and plant regeneration | |
| CN104497120B (en) | The application of grouper Clonorchiasis Sinensis TCTP and its encoding gene in terms of anti-fish nervous necrosis virus | |
| Sidyakina et al. | Viability and genetic stability of the bacterium Escherichia coli HB101 with the recombinant plasmid during preservation by various methods | |
| CN104059938B (en) | The rna interference vector of citrus fruit fly paravoltage dependent sodium channel gene and construction method thereof and application | |
| CN105950637A (en) | Trehalose-6-phosphate synthase gene of Litopenaeus vannamei and detection and application of trehalose-6-phosphate synthase gene of Litopenaeus vannamei | |
| Jo et al. | Cryopreservation of sporothalli of the genus Porphyra (Bangiales, Rhodophyta) from Korea | |
| CN107155446B (en) | Method for promoting germination of mallotus nepalensis seeds after low-temperature storage | |
| CN106613838B (en) | A method of improving regeneration rate after arabidopsis stem apex cryopreservation | |
| CN104059931A (en) | RNA interference vector of bactrocera dorsalis cytochrome P450 genes and construction method and application thereof | |
| MalekZadeh et al. | Cryopreservation of the axial meristem of Crocus sativus L | |
| CN103918556A (en) | Organic subculture preservation method for dioscorea bulbifera embryogenic callus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |