CN110982698A - Method for freezing and preserving haematococcus pluvialis by using dimethyl sulfoxide - Google Patents
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- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 description 6
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
本发明公开了一种利用二甲亚砜冷冻保藏雨生红球藻的方法，使用二甲亚砜(DMSO)作为冷冻保护剂对雨生红球藻进行冷冻保藏，选取特定生长阶段的藻体，并通过一系列降温操作作为冷冻保藏的前置过程。结果表明，本发明方法能够使藻体细胞质玻璃化，提高藻体抗冻能力，并有效提高藻种复苏率，在冷冻保藏1年时，藻体复苏率可达61.5％。
The invention discloses a method for freezing and preserving Haematococcus pluvialis by using dimethyl sulfoxide. Dimethyl sulfoxide (DMSO) is used as a cryoprotectant to freeze and preserve Haematococcus pluvialis, and algal bodies at a specific growth stage are selected. , and through a series of cooling operations as the pre-process of cryopreservation. The results show that the method of the invention can vitrify the cytoplasm of the algal body, improve the antifreeze ability of the algal body, and effectively improve the recovery rate of the algal species.
Description
Technical Field
The invention relates to a method for freezing and preserving haematococcus pluvialis by using dimethyl sulfoxide.
Background
Haematococcus pluvialis, also known as Haematococcus lake or Haematococcus lake, is a green alga, freshwater unicellular green alga, commonly found in nature, belonging to the order Volvocales, the family Haematococcus. The algae can propagate rapidly in the environment suitable for growth and widely exist in nature. Currently, Haematococcus pluvialis is considered one of the best organisms in nature to produce natural astaxanthin. Therefore, the industry for extracting astaxanthin from haematococcus pluvialis has wide market and prospect.
Haematococcus pluvialis is a unicellular green alga belonging to the phylum Chlorophyta, class Chlorophyceae, order Volvocales, family Haematococcus. The cell diameter is 5-50 μm. Haematococcus pluvialis is a unicellular organism, but has a complex and diverse life history. Under the condition of proper growth environment, the algae cells exist in a green swimming cell form (two flagella), when the growth environment is converted into a stress state, the flagella of the algae cells fall off and become green immobile cells, and when the growth environment continues to be deteriorated, the algae cells begin to accumulate astaxanthin and convert to the acinetospore, and finally become the acinetospore containing a large amount of astaxanthin. However, when the external environmental conditions are improved, i.e., the external environment is suitable for the growth of algal cells, the acinetospores turn into green motile cells. And before the thick-walled spores are initially formed and the astaxanthin begins to accumulate, intracellular metabolites begin to be complex, and flagella shrink and activity capability are weakened, so that the method is favorable for low-temperature preservation.
The different permeabilities of the cryoprotectants affect the mechanism by which they exert their protective effect. Osmotic cryoprotectants make cell membranes more plastic and they can bind intracellular water, prevent excessive dehydration, reduce salt toxicity and prevent large ice crystals from forming within the cell, forming a fine crystalline ice structure, which can form a gel-type glassy phase below the eutectic point, thus preventing hypertonic damage to the cells and surface damage caused by NaCl.
The method for preserving and cooling algae seeds at low temperature mainly adopts a one-step freezing method in the early stage, and the method is easy to damage the algae in the cooling process, so that the survival rate after recovery is greatly reduced. Patent publication No. CN107058110A proposes a method for preserving concentrated algae paste at-20 deg.C after cold storage and acclimatization, which can preserve a large amount of algae seeds for production to help pass through winter, but does not add cryoprotectant, and has great damage to algae cells. Patent publication No. CN105779292A proposes a method for sterilizing and preserving Haematococcus pluvialis at low temperature by two steps, which utilizes various antibiotics to inhibit and kill bacteria growing with the Haematococcus pluvialis, uses glycerol as a protective agent, and stores the bacteria at-80 ℃ after pre-freezing at-30 to-60 ℃, but the glycerol as the protective agent can be directly frozen when pre-freezing at the temperature, so that the cytoplasm of the Haematococcus pluvialis is difficult to vitrify and the Haematococcus pluvialis is easy to damage. Patent publication No. CN106755250A proposes a two-step method for preserving algae by using green algae cells, which comprises pre-freezing at 4 deg.C, adding protective agent, and freezing at-20 deg.C for preservation.
The freezing preservation technology of haematococcus pluvialis has a certain basis, but the preservation time is short, flagella fall off easily occurs once long-term storage is involved, the recovery rate of the algal body is low, the problems of slow growth and the like after recovery are solved, the thalli metabolism can not be prevented by normal-temperature storage or cold storage, the genetic drift can occur, and a series of problems such as degeneration and the like are to be solved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for freezing and preserving haematococcus pluvialis by using dimethyl sulfoxide, and solves the problem of the decline of the activity of algae in the process of preserving the seeds for a long time in the background technology.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for freezing and preserving haematococcus pluvialis by using dimethyl sulfoxide is provided, wherein stable early-stage alga bodies which do not synthesize a large amount of astaxanthin but have contracted flagella are selected as alga seeds, and the dimethyl sulfoxide is used as a freezing protective agent to be matched with a temperature reduction program to preserve the haematococcus pluvialis for a long time.
The invention specifically comprises the following steps:
1) preparation of the algae to be preserved: culturing haematococcus pluvialis to a stable early stage, wherein the cell density is 300-350/mu L before chlamydospores are formed, so as to obtain an algal culture solution to be preserved;
2) pre-cooling: adding DMSO into the culture solution of the algae to be preserved to enable the concentration of the DMSO to be 5-25%, carrying out program cooling to-0.5-5.0 ℃, maintaining the temperature range for 8-12 h, and carrying out precooling to obtain a seed preservation system containing slowly dehydrated cytoplasmic vitrified algae;
3) and (4) preservation: and (4) carrying out programmed cooling on the pre-cooled seed preservation system to-80 ℃, and maintaining the temperature for freezing preservation, wherein the preservation period is 1-2 years.
In a preferred embodiment of the invention, in the step 1), haematococcus pluvialis is inoculated into a BBM culture medium, and cultured under the conditions of illumination of 1500lx and light-dark ratio of 12h:12h to reach a stable early stage, and before chlamydospores are formed, an algae culture solution to be preserved is obtained;
3. the method of claim 1, further comprising the step of freezing and preserving H.pluvialis with dimethyl sulfoxide:
4) and (3) recovering the algae: thermally shocking the preserved algae in hot water at 40 deg.C, rapidly thawing to ice-free state, centrifuging to remove supernatant containing DMSO, resuspending with fresh BBM culture medium, inoculating into BBM culture medium, and culturing under illumination of 1500lx and light-dark ratio of 12h:12 h.
In a preferred embodiment of the present invention, in the step 2), the concentration of DMSO in the culture solution is 5 to 6%, the temperature is reduced to-0.5 ℃ by a procedure with a cooling rate of 0.2 to 0.5 ℃/min, and the temperature is maintained within the range of 10 to 12 hours for pre-cooling.
In a preferred embodiment of the present invention, in the step 3), the pre-cooled seed preservation system is cooled to-80 ℃ by a program with a cooling rate of 0.5 ℃/min, and is kept at the temperature for freezing preservation, and the preservation period is 1 year.
In a preferred embodiment of the present invention, the recovery rate of the algae is not less than 60%.
Compared with the background technology, the technical scheme has the following advantages:
the invention uses dimethyl sulfoxide (DMSO) as a freezing protective agent to carry out freezing preservation on haematococcus pluvialis, selects algae in a specific growth stage, firstly adds the protective agent into the algae, then leads the algae to be pre-frozen to a certain temperature by controlling the cooling rate, then stands for a period of time at the temperature, and then puts into an ultra-low temperature environment for preservation. Because the cooling rate is strictly controlled, the algae adapts to the low-temperature environment in the gradual cooling process, the damage to the algae in the cooling process is effectively reduced (as shown in figure 1), and the survival rate of the algae after recovery is greatly improved. The haematococcus pluvialis subjected to freezing preservation by the method has a preservation period of 1-2 years, a recovery rate of 61.5%, the content of chlorophyll and carotenoid after recovery is within +/-5% of the content of original algae seeds, and the yield of the algae seeds is not reduced; observing the cultured algae cells with a microscope, wherein the activity of the recovered algae is equivalent to that before preservation, the algae is full and the swimming speed is high.
Drawings
FIG. 1 is a Haematococcus pluvialis growth curve;
FIG. 2 is a schematic flow chart of the method of the present invention and a conventional one-step process;
FIG. 3 is a photomicrograph of the morphology of algae, wherein a is precooling and b is recovery culture to 14 days;
FIG. 4 is a curved surface graph of a response surface, wherein A is a seed period and B is a protective agent concentration;
FIG. 5 is a curved view of the response surface, where C is the pretreatment temperature and D is the pretreatment time.
Detailed Description
Example 1
Referring to fig. 2, the method for freezing and preserving haematococcus pluvialis by using dimethyl sulfoxide of the embodiment comprises the following steps:
1) preparing a culture solution of the algae to be preserved: inoculating 25mL of algae liquid from a subculture algae seed into three glass shake flasks containing 500mL of BBM culture liquid (inoculum size is 5%), culturing for 75h at the illumination of 1500lx and the light-dark ratio of 12h:12h and at the temperature of 22 ℃, taking the algae liquid which is cultured for 41d to a stable earlier stage and before the chlamydospores are formed, and taking the algae liquid as an algae culture liquid to be preserved, wherein the cell density is 300-350/[ mu ] L;
2) pre-cooling: adding DMSO into the culture solution of the alga to be preserved to make the concentration of the DMSO 5.3%, transferring the alga to a programmed cooling instrument at 25 ℃, setting a cooling program to be 0.2 ℃/min, cooling to-0.5 ℃, pre-cooling for 10.6h at-0.5 ℃, sampling and observing under a microscope to obtain a seed preservation system containing slowly dehydrated cytoplasmic vitrified alga;
3) and (4) preservation: setting the temperature reduction program of the pre-cooled seed preservation system to be 0.5 ℃/min, carrying out program temperature reduction to-80 ℃, maintaining the temperature, transferring to a low-temperature refrigerator for freezing preservation, and preserving for 1 year.
4) And (3) recovering the algae: thermally shocking the preserved algae in hot water at 40 deg.C, rapidly thawing until there is no ice, centrifuging to remove DMSO, inoculating to BBM culture medium, resuspending, culturing under illumination of 1500lx and light-dark ratio of 12h:12h, counting cells at 14d, and measuring chlorophyll and carotenoid and activity.
Firstly, measuring the recovery rate of algae:
culturing the seed liquid after heat shock melting according to the inoculation density and culture conditions of the culture liquid before seed preservation, counting algae cells after culturing for 14 days, and calculating the recovery rate of the seed liquid with the cell density of the culture liquid before seed preservation being 100% when the culture liquid is cultured for 14 days; extracting chlorophyll and carotenoid from Haematococcus pluvialis by acetone grinding extraction, measuring absorbances of the Haematococcus pluvialis at wavelengths of 662nm, 644nm and 440nm, respectively, and comparing, wherein when the content of the chlorophyll and the carotenoid is within +/-5% of the original content of the algae, the algae is considered to have no capacity decline; observing cultured algae somatic cells with a microscope, wherein the observation period is provided with mobility, the degree of free displacement is used as an evaluation standard of the activity of algae seeds, and the activity of original algae seeds is measured at 0.05mm/s below a blood counting cell plate.
Through counting and detection, the recovery rate of the algae is 61.5%, and the contents of chlorophyll and carotenoid in three wavelengths are respectively 96.3%, 103.5% and 101.9% of the original strain, which indicates that the accumulation of the pigments in the algae is normal and the production capacity is not obviously declined.
The morphology of the algal bodies cultured to 14d in the precooling and the recovery are shown as a and b in FIG. 3, respectively, by microscopic observation. After precooling, partial dehydration appears in the interior of the alga body, and cytoplasm is concentrated; the activity of the recovered algae is equivalent to that before preservation, and the algae is full and fast in swimming speed.
Second, freezing preservation response surface experiment
1) Inoculating 25mL of algal solution into three glass shake flasks in 500mL of BBM culture solution (inoculum size is 5%) from the subculture algal seeds, culturing for 75h at the illumination of 1500lx and the light-dark ratio of 12h:12h and at the temperature of 22 ℃, taking cultured 9d, 27d and 45d as different seed stages for preservation experiments, and measuring the growth curves as shown in figure 1.
2) Selecting 3 different seed stages, using the concentration of dimethyl sulfoxide, the precooling temperature and the precooling time as influence factors to carry out a response surface optimization experiment, carrying out a 4-factor 3 horizontal response surface experiment, preserving at-80 ℃ for six months and investigating the survival rate of the recovered algae, wherein the specific level, the experimental design and the result are shown in tables 1 and 2, wherein the program cooling instrument sets the cooling speed to be 0.5 ℃/min:
table 1 experimental horizontal design
Table 2 experimental design and results
The following equation is obtained by analyzing with Design Expert software
R1＝0.17126+0.078630×A-0.057335×B-0.046739×C+0.019378×D-3.58929E-003×A×B-3.41270E-003×A×C+0.028968×A×D+0.071181×B×C+2.43056E-003×B×D-0.037847×C×D-0.033441×A2-0.073650×B2-0.017708×C2-0.046954×D2
Wherein the R1 value is a response value (i.e. algal species resuscitation rate), A is a seed period, B is a protective agent concentration, C is a pretreatment temperature, D is a pretreatment time, A, B, C, D is 4 analytical factors, and the significance of the model and each parameter is verified by analysis of variance (ANOVA). From side to sideThe parameters A-A, B-B, C-C, B in the model can be seen by difference analysis2Has obvious influence on the recovery rate of algae (Prob)>F is less than 0.05), F is 2.73, which indicates that the model has a good degree of fit and a high degree of confidence, and therefore, the change in the response value can be analyzed using this model.
From the results, it can be seen that the seed stage has the most significant effect on the algal resuscitation rate, while the protectant concentration and pretreatment temperature effects are more significant, while the pretreatment time effects are not significant. Within the influence range of each selected factor, the influence on the resuscitation rate is ranked, and the seed stage > protective agent concentration > pretreatment temperature > pretreatment time.
TABLE 3 results of regression analysis
The response surface curve diagrams are shown in fig. 4 and 5, and it can be seen that the response center determined by the experiment is closer to the highest point predicted by the model, which shows that the single-factor experiment has a very important role in determining the concentration range of each factor. Because the variety of the factors to be screened is more, the variety and the approximate concentration range of the optimized factors can be more conveniently determined by using multi-factor screening and single-factor concentration gradient experiments to investigate the mutual influence among the screened factors, thereby determining the optimum seed-protecting condition.
The regression equation is further analyzed by using Design Expert, and the obtained solution is that a is 0.81, B is-0.97, C is-0.95, D is 0.78, which are maximum points of the curved surface, the optimal conditions of the corresponding factors are that the concentration of the protective agent is 5.3% for the seed growth time 41.58D, the pretreatment temperature is-0.5 ℃, and the pretreatment time is 10.68 h. The theoretical recovery rate under the condition is the highest and is 66.1%.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
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| 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 |
| CN106755250A (en) * | 2016-12-27 | 2017-05-31 | 山东金晶生物技术有限公司 | A kind of preservation of haematococcus pluvialis green cell and the large-scale method for producing of astaxanthin induction |
| CN107058110A (en) * | 2017-07-04 | 2017-08-18 | 中国科学院青岛生物能源与过程研究所 | A kind of preservation of haematococcus pluvialis cell and method for resuscitation |
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|---|---|---|---|---|
| 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 |
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| 廖万清: "《现代真菌病学》", 31 October 2017 * |
| 盘赛昆等: "鳙鳃凝集素的稳定性及细胞凝集作用 ", 《安徽农业科学》 * |
| 郑凌凌等: "雨生红球藻FACHB-712 营养细胞和厚壁孢子低温保藏效果及优化", 《植物科学学报》 * |
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