Application of chrysophyceae culture supernatant in chrysophyceae preservation

Abstract

The invention relates to the field of algae preservation, in particular to application of supernate in chrysophyceae preservation, a chrysophyceae preservation method and application of the chrysophyceae preservation method. The invention discloses application of a chrysophyceae culture supernatant and an additive in prolonging the freezing shelf life of chrysophyceae, reducing clotting after freezing and thawing or reducing nutrient loss after freezing and thawing. The additive is glycerol and / or vitamin. The invention further discloses a method for preserving the chrysophyceae, the method comprises the step that the chrysophyceae preserving fluid is subjected to cryopreservation, the chrysophyceae preserving fluid comprises the chrysophyceae, the additive and the chrysophyceae culture supernatant, and the additive is glycerinum and / or vitamin. According to the cryopreservation method of the chrysophyceae, the preservation time of the chrysophyceae can be prolonged, after freeze thawing, the cell structure is complete, uniform in dispersion and free of obvious caking, nutritional ingredients are not prone to loss, and the cryopreservation method can be used for breeding spats and shrimp seeds.

Description

Technical Field

[0001] The present invention relates to the field of algal preservation, and particularly to the application of supernatant in the preservation of Chrysochromulina, a method for preserving Chrysochromulina, and its application. Background Art

[0002] Microalgae are tiny and rich in nutrients, and are excellent natural baits for aquatic animals. Some marine species have been artificially cultured and are important baits during the artificial breeding of aquaculture economic animals. In current breeding production, the cost of breeding often increases or even breeding fails due to untimely supply, insufficient supply, and single variety of fresh bait microalgae. Therefore, it is necessary to reserve a certain amount of concentrated algal liquid or algal paste for use when fresh algal liquid is lacking.

[0003] Both low-temperature preservation and cryopreservation are common means for preserving microalgae concentrates. CN105838611A proposes a method for preserving Chlorella vulgaris concentrate, which uses flocculation and centrifugation methods to prepare Chlorella vulgaris concentrate, and adds a combination of one or two cryoprotectants of dimethyl sulfoxide and methanol when cryopreserving at -10°C and -30°C, and the preservation time is 3 months. However, both dimethyl sulfoxide and methanol have certain toxicity, and it is difficult to ensure product safety when added to the concentrated algal liquid, so it is difficult to be practically applied in aquaculture.

[0004] CN105779293A proposes a method for preserving Chlorella vulgaris, adding a compound agent with a concentration of 2% glycerol, 0.06% potassium sorbate, and 2% tea extract concentration to the Chlorella vulgaris concentrate. After storing at 28°C for 30 days, the loss of each nutrient component is less, but the cell concentration of the Chlorella vulgaris concentrate is too low and the storage time is too short to be applied in large-scale aquaculture; potassium sorbate in the additives used in this method is toxic, and it is difficult to ensure safety in aquaculture. Moreover, the types of additives in this method are numerous and the preparation is complex.

[0005] Freezing is also a common preservation method. However, when preserving concentrated algal liquid under freezing conditions, a large amount of agglomeration will occur in algal cells after freeze-thawing, which is not applicable to the breeding of shellfish and shrimp. Chrysochromulina is widely used in aquaculture and is the main first-feeding bait for the breeding of shellfish and shrimp. The diameter of the first-feeding bait for shellfish larvae and shrimp larvae should be less than 20 μm. Therefore, in practical applications, if Chrysochromulina cells agglomerate, they cannot be utilized by the larvae.

[0006] In addition, existing concentrated algal liquids are all cultured on a small scale in the laboratory, mainly used for the preservation and resurrection culture of algal species, and are difficult to be directly applied in aquaculture.

[0007] Therefore, there is an urgent need for a cryopreservation method for Chrysochromulina with a long storage time, small nutrient loss after freeze-thawing, and not easy to agglomerate. Summary of the Invention

[0008] The object of the present invention is to overcome the problems in the prior art that the shelf life of frozen preservation of chrysophytes is short and agglomeration easily occurs after freeze-thawing, and to provide an application of supernatant in the preservation of chrysophytes, a method for preserving chrysophytes and its application.

[0009] In the current methods for preserving chrysophytes, generally, the culture supernatant in the chrysophyte culture solution is first removed, and then the collected chrysophyte thalli are mixed with cryoprotectants and the like for frozen preservation. However, the inventors of the present invention unexpectedly found in the research that using the chrysophyte culture supernatant for frozen preservation of chrysophytes can well avoid the problems of short shelf life and easy agglomeration after freeze-thawing. Therefore, in order to achieve the above object, the first aspect of the present invention provides an application of a chrysophyte culture supernatant and an additive in improving the frozen shelf life of chrysophytes, reducing agglomeration after freeze-thawing or reducing the loss of nutritional components after freeze-thawing, and the additive is glycerol and / or vitamin.

[0010] The second aspect of the present invention provides a method for preserving chrysophytes, wherein the method includes freezing and preserving a chrysophyte preservation solution;

[0011] wherein, the chrysophyte preservation solution contains chrysophytes, an additive and the chrysophyte culture supernatant described in the first aspect of the present invention, and the additive is glycerol and / or vitamin.

[0012] The third aspect of the present invention provides a method for aquaculture, wherein the method includes:

[0013] (1) Preserving chrysophytes according to the method described in the second aspect of the present application, and thawing the frozen preservation;

[0014] (2) Feeding the thawed product obtained in step (1) to aquaculture animals.

[0015] Through the above technical solutions, the present invention has at least the following beneficial effects:

[0016] (1) The method for frozen preservation of chrysophytes described in the present invention is simple in operation, can improve the preservation time of chrysophytes, and after freeze-thawing, the cell structure is complete, evenly dispersed, without obvious agglomeration, and the nutritional components are not easily lost, and can be used for the breeding of shellfish seedlings and shrimp seedlings.

[0017] (2) The other components added to the chrysophyte preservation solution are simple, which not only ensures the preservation effect, but also ensures the safety of the application of chrysophytes in aquaculture, and does not affect the nutritional value of chrysophytes, so that the chrysophyte preservation solution can be directly used as bait in aquaculture. Description of the Drawings

[0018] Figure 1 It is the microscopic examination result of the cell state of the chrysophyte concentrate after being preserved at different temperatures for 90 days. a is 4°C; b is -20°C.

[0019] Figure 2are the cell densities of the Isochrysis galbana preservation solutions obtained from the control group and experimental groups 1-3 after being cryopreserved at -20°C for different days.

[0020] Figure 3 are the microscopic examination results of the cell states of the Isochrysis galbana preservation solutions obtained from the control group and experimental groups 1-3 after being cryopreserved at -20°C for 90 days: a is the control group; b is experimental group 1; c is experimental group 2; d is experimental group 3.

[0021] Figure 4 are the microscopic examination results of the cell states of the Isochrysis galbana preservation solutions obtained from the control group and experimental groups 1-3 after being cryopreserved at -20°C for 180 days: a is the control group; b is experimental group 1; c is experimental group 2; d is experimental group 3.

[0022] Figure 5 are the contents of the main nutrients of the algal powder obtained by vacuum freeze-drying the Isochrysis galbana preservation solutions obtained from the control group and experimental groups 1-3 after being cryopreserved at -20°C for 0 day, 10 days, 30 days, 90 days, and 180 days. Specific Embodiments

[0023] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0024] The first aspect of the present invention provides an application of an Isochrysis galbana culture supernatant and an additive in improving the frozen shelf life of Isochrysis galbana, reducing agglomeration after freeze-thawing, or reducing the loss of nutrients after freeze-thawing, and the additive is glycerol and / or vitamin.

[0025] In the present invention, the addition amount of the glycerol, the type and addition amount of the vitamin are as described in the second aspect of the present invention.

[0026] In the present invention, preferably, the preparation method of the Isochrysis galbana culture supernatant includes inoculating Isochrysis galbana into a liquid medium for Isochrysis galbana culture, and then collecting the supernatant (removing the algal bodies) from the Isochrysis galbana culture solution.

[0027] In the present invention, the conditions for Isochrysis galbana culture can enable the normal growth of Isochrysis galbana. In some embodiments of the present invention, in order to enable better growth of Isochrysis galbana, preferably, the conditions for Isochrysis galbana culture include: the inoculation amount of Isochrysis galbana makes the initial OD of the algal solution 680 be 0.3 - 0.6.

[0028] In some embodiments of the present invention, in order to enable better growth of Isochrysis galbana, preferably, the culture pH for Isochrysis galbana culture is 6 - 8.

[0029] In some embodiments of the present invention, in order to enable better growth of the chrysophyte, preferably, the temperature of the algal solution for culturing the chrysophyte is 25 - 35 °C.

[0030] In some embodiments of the present invention, in order to enable better growth of the chrysophyte, preferably, the light intensity for culturing the chrysophyte is 20 - 50 μmol·m -2 ·s -1 。

[0031] In the present invention, the chrysophyte can be cultured until the stationary phase. According to some preferred embodiments of the present invention, the culturing time of the chrysophyte is 6 - 10 days.

[0032] In some embodiments of the present invention, in order to enable better growth of the chrysophyte, preferably, the salinity of the liquid medium is 1% - 4%, more preferably 2% - 3%. "Salinity" refers to the mass fraction of salts in seawater and can be measured by a salinometer method.

[0033] In some embodiments of the present invention, in order to enable better growth of the chrysophyte, preferably, the liquid medium is a modified f / 2 medium.

[0034] In some embodiments of the present invention, more preferably, the modified f / 2 medium contains 0.5 - 5 g / L NaNO 3 ,50 - 70 mg / L NaH 2 PO 4 ,1 - 10 mg / L FeCl 3 ,5 - 50 μg / L CuSO 4 ,15 - 50 μg / L ZnSO 4 ,50 - 400 μg / L MnCl 2 ,5 - 50 μg / L Na 2 MoO 4 ,2 - 20 μg / L CoCl 2 ,1 - 20 μg / L vitamin B 12 ,70 - 120 μg / L vitamin B 1 ,1 - 20 μg / L biotin, 10 - 50 g / L sea salt.

[0035] In some embodiments of the present invention, further preferably, the modified f / 2 medium contains 0.5 - 1.5 g / L NaNO 3 ,55 - 60 mg / L NaH 2 PO 4 ,1 - 5 mg / L FeCl 3 ,5 - 20 μg / L CuSO 4, 20 - 30 μg / L ZnSO 4 , 200 - 300 μg / L MnCl 2 , 5 - 15 μg / L Na 2 MoO 4 , 2 - 10 μg / L CoCl 2 , 3 - 8 μg / L vitamin B 12 , 90 - 110 μg / L vitamin B 1 , 1 - 10 μg / L biotin, 20 - 30 g / L sea salt.

[0036] In the present invention, preferably, the collection method is concentration, and the concentration method can be a conventional method in the art without particular limitation. For example, it can be at least one of centrifugation and filtration.

[0037] In the present invention, preferably, the concentration is centrifugation.

[0038] In the present invention, the supernatant can be the supernatant after each centrifugation or the supernatant obtained by combining the supernatants after any number of centrifugations.

[0039] In the present invention, in order to reduce the damage to the chrysophyta cells and extend the frozen shelf life, preferably, the centrifugation is at least two times of centrifugation.

[0040] In the present invention, in order to reduce the damage to the chrysophyta cells and extend the frozen shelf life, preferably, in the at least two times of centrifugation, the speed of each centrifugation is 3000 - 8000 rpm / min, for example, it can be 3000 rpm / min, 4000 rpm / min, 5000 rpm / min, 6000 rpm / min, 7000 rpm / min, 8000 rpm / min and any range composed of any two of these values, and more preferably 4000 - 6000 rpm / min.

[0041] In the present invention, in order to reduce the damage to the chrysophyta cells and extend the frozen shelf life, preferably, in the at least two times of centrifugation, the time of each centrifugation is 5 - 20 min, for example, it can be 5 min, 8 min, 10 min, 13 min, 15 min, 18 min, 20 min and any range composed of any two of these values, and more preferably 5 - 15 min.

[0042] In the present invention, in order to further reduce the damage to the chrysophyta cells and extend the frozen shelf life, in the at least two times of centrifugation, the speed of the first centrifugation is 3000 - 8000 rpm / min, preferably 4000 - 6000 rpm / min; the time of the first centrifugation is 5 - 20 min, preferably 5 - 15 min.

[0043] The inventors of the present invention have found that, in the at least two centrifugations, when the speed of the second centrifugation is lower than that of the first centrifugation, the damage to the chrysophyta cells can be further reduced and the frozen shelf life can be extended. Preferably, the speed of the second centrifugation is 50 - 500 rpm / min lower than that of the first centrifugation. For example, it can be 50 rpm / min lower, 100 rpm / min lower, 150 rpm / min lower, 200 rpm / min lower, 250 rpm / min lower, 300 rpm / min lower, 350 rpm / min lower, 400 rpm / min lower, 450 rpm / min lower, 500 rpm / min lower, and any range composed of any two of these values. More preferably, it is 150 - 300 rpm / min lower.

[0044] The second aspect of the present invention provides a method for preserving chrysophyta, wherein the method includes cryopreserving the chrysophyta preservation solution;

[0045] Wherein, the chrysophyta preservation solution contains chrysophyta, an additive, and the chrysophyta culture supernatant described in the first aspect of the present invention, and the additive is glycerol and / or vitamin.

[0046] The present invention can directly cryopreserve the chrysophyta culture solution, or can concentrate the chrysophyta culture solution (which can be obtained by inoculating chrysophyta into a liquid medium for chrysophyta culture) and then cryopreserve it.

[0047] In the present invention, the cell density of chrysophyta in the chrysophyta preservation solution is not particularly limited. To increase the number of preserved chrysophyta cells and reduce the preservation cost, preferably, the cell density of chrysophyta in the chrysophyta preservation solution is 1×10 9 -1×10 10 cells / mL. For example, it can be 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 and any range composed of any two of these values. More preferably, it is 3×10 9 -6×10 9 cells / mL.

[0048] In the present invention, in order to further extend the storage time, reduce agglomeration after freeze-thawing and reduce nutrient loss after freeze-thawing, preferably, the chrysophyte includes at least one selected from Isochrysis, Chromulina, and Ochromonas, for example, it can be Isochrysis galbana or Isochrysis zhanjiangensis in Isochrysis; Chromulina tropica or Chromulina ovalis in Chromulina; Ochromonas mobilis, Ochromonas mutabilis or Ochromonas vallescia in Ochromonas.

[0049] In the present invention, preferably, the storage temperature is -10°C to -80°C, for example, it can be -10°C, -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C and any range composed of any two of these values. In order to further extend the storage time, reduce agglomeration after freeze-thawing and reduce nutrient loss after freeze-thawing, the storage temperature is more preferably -10°C to -30°C.

[0050] In the present invention, preferably, the cryopreservation time is 0 - 360 days, for example, it can be 0 days, 10 days, 20 days, 30 days, 60 days, 90 days, 120 days, 150 days, 180 days, 210 days, 240 days, 270 days, 300 days, 330 days, 360 days and any range composed of any two of these values. In order to further reduce agglomeration after freeze-thawing and reduce nutrient loss after freeze-thawing, the cryopreservation time is more preferably 0 - 180 days.

[0051] In large-scale aquaculture, the shelf life of microalgae can meet the application requirements when it is 90 days. The inventors of the present invention found that by using some of the preferred preservation methods of the invention, for chrysophytes stored for 180 days, the algal cells are still evenly distributed after freeze-thawing, not easily agglomerated, and the loss of nutrient components of the algal cells is small. Moreover, according to actual usage requirements, the storage time can be further extended, for example, extended to 360 days.

[0052] In the present invention, in order to reduce the toxicity to algal cells and improve the safety of chrysophytes in aquaculture applications, preferably, the chrysophyte preservation solution does not contain an externally added cryoprotectant.

[0053] In the present invention, in order to extend the storage time, reduce agglomeration after freeze-thawing and reduce nutrient loss after freeze-thawing, preferably, the chrysophyte preservation solution further includes additives, and the additives are glycerol and vitamins.

[0054] In the present invention, the preparation of the chrysophyte preservation solution can be to directly add additives to the non-concentrated chrysophyte culture solution; or to concentrate the chrysophyte culture solution to the required cell concentration and then add additives, or add additives and supernatant; or to concentrate the chrysophyte culture solution into a paste-like algal mud and then add supernatant and additives.

[0055] In the present invention, in order to extend the storage time, reduce agglomeration after freeze-thaw cycles, and reduce nutrient loss after freeze-thaw cycles, preferably, in the Isochrysis storage solution, for every 10 billion Isochrysis cells, the addition amount of glycerol is 50 - 500 mg, for example, it can be 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, and any range composed of any two of these values. In order to further extend the storage time, reduce agglomeration after freeze-thaw cycles, and reduce nutrient loss after freeze-thaw cycles, in the Isochrysis storage solution, for every 10 billion Isochrysis cells, the addition amount of glycerol is more preferably 50 - 350 mg.

[0056] In the present invention, in order to extend the storage time, reduce agglomeration after freeze-thaw cycles, and reduce nutrient loss after freeze-thaw cycles, preferably, the vitamin includes vitamin B 1 , vitamin B 12 and biotin.

[0057] In the present invention, in order to extend the storage time, reduce agglomeration after freeze-thaw cycles, and reduce nutrient loss after freeze-thaw cycles, preferably, based on the volume of the Isochrysis storage solution, the concentration of vitamin B 1 is 20 - 200 μg / L, more preferably 50 - 150 μg / L.

[0058] In the present invention, in order to extend the storage time, reduce agglomeration after freeze-thaw cycles, and reduce nutrient loss after freeze-thaw cycles, preferably, based on the volume of the Isochrysis storage solution, the concentration of vitamin B 12 is 1 - 20 μg / L, preferably 2 - 10 μg / L.

[0059] In the present invention, in order to extend the storage time, reduce agglomeration after freeze-thaw cycles, and reduce nutrient loss after freeze-thaw cycles, preferably, based on the volume of the Isochrysis storage solution, the concentration of biotin is 1 - 20 μg / L, preferably 2 - 10 μg / L.

[0060] In a particularly preferred embodiment of the present invention, the method for storing Isochrysis includes cryopreserving the Isochrysis storage solution; wherein, the Isochrysis storage solution contains the Isochrysis culture supernatant obtained by combining the supernatants of each centrifugation described in the first aspect of the present invention; the cell density of Isochrysis in the Isochrysis storage solution is 3×10 9 -6×10 9 cells / mL; the Isochrysis includes at least one selected from Isochrysis galbana, Chromulina sp., and Ochromonas sp.; the temperature of the cryopreservation is -10°C to -30°C; the time of the cryopreservation is 0 - 180 days; the Isochrysis storage solution does not contain an externally added cryoprotectant; the additive includes glycerol and vitamin; for every 10 billion Isochrysis cells, the addition amount of glycerol is 50 - 350 mg; the vitamin includes vitamin B1 , vitamin B 12 and biotin; based on the volume of the Isochrysis galbana preservation solution, the concentration of the vitamin B 1 is 50 - 150 μg / L; based on the volume of the Isochrysis galbana preservation solution, the concentration of the vitamin B 12 is 2 - 10 μg / L; based on the volume of the Isochrysis galbana preservation solution, the concentration of the biotin is 2 - 10 μg / L.

[0061] The third aspect of the present invention provides a method for aquaculture, which includes:

[0062] (1) Preserve Isochrysis galbana according to the method described in the second aspect of the present application, and thaw the frozen preservation;

[0063] (2) Feed the thawed product obtained in step (1) to aquaculture animals.

[0064] In the present invention, the method for aquaculture can be to directly feed the thawed Isochrysis galbana preservation solution to aquaculture animals, such as shrimp or shellfish larvae; or the thawed Isochrysis galbana preservation solution can be processed as needed and then used for aquaculture. For example, it can be concentrated to obtain Isochrysis galbana concentrated algal solution, Isochrysis galbana concentrated algal paste or Isochrysis galbana algal powder, and then used for aquaculture.

[0065] The present invention will be described in detail below through examples.

[0066] In the following preparatory examples, experimental groups and control groups, the Isochrysis galbana used is Isochrysis galbana, preserved in the Freshwater Algae Culture Collection of the Chinese Academy of Sciences, and the algal strain number is FACHB - 861.

[0067] The cell density of microalgae was measured by the hemocytometer counting method.

[0068] The sea salt was purchased from Zhejiang Blue Sea Star Salt Products Co., Ltd.

[0069] The salinity of the culture medium was measured by a salinometer.

[0070] Preparatory Example

[0071] This preparatory example is used to illustrate the preparation of the supernatant.

[0072] (1) Isochrysis galbana culture: Isochrysis galbana was cultured on a large scale using a glass pipeline type photobioreactor. The culture medium was modified f / 2 medium. The initial OD of the algal solution after inoculation 680 was 0.5. Carbon dioxide gas was introduced to control the pH value at 6 - 8, the culture temperature was 25 - 30 °C, and the light intensity was controlled at 20 - 50 μmol·m -2 ·s -1, cultured for 8 days until the stationary phase, the salinity of the medium is 20‰, and the solvent is secondary water.

[0073] The f / 2 medium composition is as follows:

[0074]

[0075]

[0076] (2) Centrifuge the obtained Isochrysis galbana culture solution at 5000 rpm / min for 10 minutes, remove the supernatant to obtain the first centrifugal concentrate, and then centrifuge the first centrifugal concentrate at 4750 rpm / min for 10 minutes, remove the supernatant to obtain the Isochrysis galbana concentrate. The cell density of the Isochrysis galbana concentrate is 1×10 10 cells / mL, and the mass of each milliliter of Isochrysis galbana concentrate is about 1 g.

[0077] Experimental group 1

[0078] Add glycerol, vitamin B 1 , vitamin B 12 , and biotin to the Isochrysis galbana concentrate, and finally supplement the supernatant obtained from the first centrifugation to obtain the Isochrysis galbana preservation solution, so that the cell density of Isochrysis galbana in the Isochrysis galbana preservation solution is 3.5 billion cells / mL;

[0079] Based on the mass of the Isochrysis galbana concentrate, the content of glycerol is 10 wt%; based on the volume of the Isochrysis galbana preservation solution, the content of vitamin B 1 is 100 μg / L, the content of vitamin B 12 is 5 μg / L, and the content of biotin is 5 μg / L.

[0080] Experimental group 2

[0081] The preservation method is the same as that of experimental group 1. The difference is that based on the mass of the Isochrysis galbana concentrate, the content of glycerol is 20 wt%; based on the volume of the Isochrysis galbana preservation solution, the content of vitamin B 1 is 100 μg / L, the content of vitamin B 12 is 5 μg / L, and the content of biotin is 5 μg / L.

[0082] Experimental group 3

[0083] The preservation method is the same as that of experimental group 1. The difference is that based on the mass of the Isochrysis galbana concentrate, the content of glycerol is 30 wt%; based on the volume of the Isochrysis galbana preservation solution, the content of vitamin B 1 is 100 μg / L, the content of vitamin B 12 is 5 μg / L, and the content of biotin is 5 μg / L.

[0084] Control group

[0085] The preservation method was the same as that of Experimental Group 1, except that only the supernatant obtained from the first centrifugation was added.

[0086] Test Example

[0087] (1) The concentrated Chlorella solution obtained in the Preliminary Example and the Chlorella preservation solutions obtained in the control group and the experimental groups were sealed and stored at 4 °C and -20 °C for observation.

[0088] After the concentrated Chlorella solution was stored at 4 °C for 90 days, white bacterial colonies appeared on the surface, the algal solution had an obvious odor, and a large number of bacteria were observed to multiply in the algal solution by microscopic examination, and the degradation of the algal cell chromatophores was obvious ( Figure 1 a). After being stored at -20 °C for 90 days and thawed, there were obvious agglomerated particles, the algal solution had no obvious peculiar smell, and the multiplication of bacteria in the algal solution was significantly inhibited by microscopic examination, the algal cells were intact and no obvious degradation occurred ( Figure 1 b).

[0089] When the Chlorella preservation solution obtained in the control group was stored at 4 °C for the same time, it became smelly and deteriorated more easily than the experimental groups.

[0090] (2) The Chlorella preservation solutions obtained in the control group and the experimental groups were sealed and stored frozen at -20 °C. During this period, samples of the same volume were taken at 0 days, 10 days, 30 days, 90 days and 180 days from the start of the experiment to observe the color, uniformity, odor, cell state of the samples, and calculate the cell density. The results are shown in Table 1 and Figure 2 .

[0091] Samples of the same volume were taken from the control group and the experimental groups at 90 days and 180 days of storage for microscopic examination. The results are shown in Figure 3 、 Figure 4 .

[0092] The uniformity refers to the uniformity of the texture of the algal solution observed with the naked eye. If there are no obvious particles (agglomerates formed by the aggregation of a large number of algal cells) in the algal solution, it is judged as uniform;

[0093] The cell state refers to the integrity and dispersion degree of the cells observed by microscopic examination. Clear cell boundaries and intact chromatophores indicate good cell integrity, unclear cell boundaries and degraded chromatophores indicate cell breakage and poor integrity. If more than 95% of the cells are individually dispersed, it is considered to be evenly dispersed, and the aggregation and adhesion of multiple cells are considered to be agglomerated (for example Figure 3 a)

[0094] In the cell density, if the cell density decreases by no more than 10%, it is judged as "not significantly decreased".

[0095] (3) On the basis of (2), samples of the same volume were taken, diluted 10 times with the modified f / 2 medium, centrifuged, the supernatant was removed, and freeze-dried under vacuum into algal powder, and the contents of the nutrient components were measured. The results are shown in Figure 5Using the same method, the cultured chlorella was made into algal powder, and the contents of nutritional components were measured. The results are shown in Table 2.

[0096] Figure 5 In Table 2, dwt refers to "Dry weight", dry weight.

[0097] Qualitative and quantitative analysis methods for algal powder pigments (including total carotenoids, fucoxanthin, chlorophyll): 1) Weigh 10 mg of algal powder into a centrifuge tube, add a certain amount of methanol, and use a Fast Nucleic Acid Extractor (Fast Prep-24, manufactured by MP bio) to break the cell wall and extract pigments. Then centrifuge at 16,000 g for 10 minutes, and pipette the supernatant extract into a new collection tube. Repeat the extraction step until the algal residue is colorless. Combine the extracts and filter the extract with a 0.22 μm filter membrane to prepare the sample for analysis. 2) Use a liquid chromatograph (model Waters 2695) and an ultraviolet detector (model Waters 2489) to qualitatively and quantitatively analyze the pigments contained in the algal powder. The chromatographic column is a Spherisorb C18 column (5 μm, 4.6 mm, 250 mm) (manufactured by Waters Corporation, MA, USA), and the column temperature is 40°C. The mobile phases are A: ethyl acetate, B: 0.1 M Tris-HCl (pH 8.0), C: acetonitrile, D: methanol, and gradient elution is performed. The absorption of chlorophyll and carotenoids (fucoxanthin, zeaxanthin, lutein, β-carotene, astaxanthin) is detected at 664 nm and 445 nm respectively, and quantitative analysis is carried out through the standard curves of chlorophyll and carotenoids (manufactured by Sigma-Aldrich, MO, USA).

[0098] Method for determining crude protein in algal powder: Weigh 10 mg of algal powder, add 100 μL of 1 M NaOH, heat in a water bath at 80°C for 10 minutes, then add 900 μL of deionized water, centrifuge at 16,000 g for 10 minutes at 4°C, collect the supernatant, and then repeat the water bath extraction step 2 times by adding 1 M NaOH to the remaining algal residue. The crude protein content is measured using a modified Bradford protein analysis kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.).

[0099] Qualitative and Quantitative Analysis Method for Algal Powder Fatty Acids (including Total Fatty Acids, DHA): Weigh 10 mg of algal powder sample, and successively add 200 μL of chloroform: methanol (2:1, v / v), 300 μL of 5% HCl: methanol solution (3:1, v / v), and 25 μL of 10 mg / mL C13:0 n-hexane solution. Shake well and place at 85 °C for 1 hour to extract and methylate the algal powder oil. After methylation, add 1 mL of n-hexane to the sample bottle, shake well, and let it stand at room temperature for 1 to 4 hours to extract fatty acids. Finally, take 200 μL of the upper-layer extract (which can be diluted with n-hexane according to the actual situation) and add 5 μL of 1 mg / mL pentadecane (internal standard) to a gas chromatography sample bottle with an inner cannula to prepare the sample for analysis. Use an Agilent gas chromatography-mass spectrometry instrument (model 7890B-5977A) for qualitative analysis of fatty acids, and use an Agilent 7890B gas chromatograph and a flame ionization detector (GC-FID) for quantitative analysis.

[0100] Table 1

[0101]

[0102]

[0103] Table 2

[0104] Nutritional components Content (dwt%) ± SD Total carotenoids 2.00±0.03 Fucoxanthin 1.60±0.01 Chlorophyll 1.75±0.02 Crude protein 40.76±0.67 Total fatty acids 13.79±0.04 DHA 1.26±0.00

[0105] It can be seen from the results in Table 1 that for experimental groups 1-3 that preserved Isochrysis galbana using supernatant, glycerol, and vitamins, after 90 days and 180 days of preservation, there were no obvious particles after freeze-thawing, the cells were intact, and evenly dispersed. Combining with Figure 2 the results shows that there was no significant decrease in cell density; the color of Isochrysis galbana in each group was dark brown, indicating that the color of the algal solution did not lighten during cryopreservation and the pigments as a whole did not degrade significantly.

[0106] The microscopic examination results of the cells after 90 days and 180 days of preservation showed that the cells in the control group agglomerated severely after thawing, while the cells in the experimental group were intact and evenly dispersed after thawing, indicating that a specific content of supernatant, glycerol, and vitamins in combination was more conducive to the long-term cryopreservation of microalgae.

[0107] Figure 5 It shows that there were no significant differences in the main nutritional components between the experimental group and the control group, indicating that adding glycerol and vitamins as cryoprotectants did not affect the main nutritional components of Isochrysis galbana.

[0108] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. Application of supernatant of Isochrysis culture and additive in improving the frozen shelf life of Isochrysis, reducing agglomeration after freeze-thawing or reducing loss of nutrients after freeze-thawing, wherein the additive is glycerol and / or vitamin.

2. The application according to claim 1, wherein, the preparation method of the supernatant of Isochrysis culture includes inoculating Isochrysis into a liquid medium for Isochrysis culture, and then collecting the supernatant from the Isochrysis culture solution; Preferably, the conditions for culturing the chrysophyte include: the inoculation amount of the chrysophyte such that the initial OD of the algal solution 680 is 0.3 - 0.6; more preferably, the culture pH of the Isochrysis culture is 6-8; more preferably, the temperature of the Isochrysis culture solution is 25-35°C; More preferably, the light intensity for culturing the chrysophyte is 20 - 50 μmol·m -2 ·s -1 ; more preferably, the culture time of the Isochrysis culture is 6-10 days; more preferably, the salinity of the liquid medium is 1%-4%, preferably 2%-3%.

3. The application according to claim 2, wherein, the liquid medium is a modified f / 2 medium; Preferably, the improved f / 2 medium contains 0.5 - 5 g / L NaNO 3 , 50 - 70 mg / L NaH 2 PO 4 , 1 - 10 mg / L FeCl 3 , 5 - 50 μg / L CuSO 4 , 15 - 50 μg / L ZnSO 4 , 50 - 400 μg / L MnCl 2 , 5 - 50 μg / L Na 2 MoO 4 , 2 - 20 μg / L CoCl 2 , 1 - 20 μg / L vitamin B 12 , 70 - 120 μg / L vitamin B 1 , 1 - 20 μg / L biotin, 10 - 50 g / L sea salt; More preferably, the improved f / 2 medium contains 0.5-1.5 g / L NaNO 3 , 55-60 mg / L NaH 2 PO 4 , 1-5 mg / L FeCl 3 , 5-20 μg / L CuSO 4 , 20-30 μg / L ZnSO 4 , 200-300 μg / L MnCl 2 , 5-15 μg / L Na 2 MoO 4 , 2-10 μg / L CoCl 2 , 3-8 μg / L vitamin B 12 , 90-110 μg / L vitamin B 1 , 1-10 μg / L biotin, 20-30 g / L sea salt.

4. The application according to claim 2 or 3, wherein, the collection method is concentration; preferably, the concentration is centrifugation and / or filtration.

5. The application according to claim 4, wherein, the centrifugation is at least two times of centrifugation; preferably, in the at least two times of centrifugation, the speed of each centrifugation is 3000-8000 rpm / min, more preferably 4000-6000 rpm / min; preferably, in the at least two times of centrifugation, the time of each centrifugation is 5-20 min, more preferably 5-15 min.

6. The application according to claim 4 or 5, wherein, in the at least two times of centrifugation, the speed of the first centrifugation is 3000-8000 rpm / min, preferably 4000-6000 rpm / min; the time of the first centrifugation is 5-20 min, preferably 5-15 min; preferably, the speed of the second centrifugation is less than that of the first centrifugation; more preferably, the speed of the second centrifugation is 50-500 rpm / min less than that of the first centrifugation, and further preferably 150-300 rpm / min less.

7. A method for preserving Isochrysis, characterized in that, the method includes freezing and preserving the Isochrysis preservation solution; wherein, the Isochrysis preservation solution contains Isochrysis, an additive and the supernatant of Isochrysis culture according to any one of claims 1-6, and the additive is glycerol and / or vitamin.

8. The method according to claim 7, wherein, The cell density of the chrysophyte in the chrysophyte preservation solution is 1×10 9 -1×10 10 cells / mL, preferably 3×10 9 -6×10 9 cells / mL; and / or, the Isochrysis includes at least one selected from Isochrysis galbana, Chromulina sp., Ochromonas sp.; preferably, the temperature of the freezing and preservation is -10°C to -80°C, preferably -10°C to -30°C; preferably, the time of the freezing and preservation is 0-360 days, preferably 0-180 days.

9. The method according to claim 7 or 8, wherein, the Isochrysis preservation solution does not contain an externally added cryoprotectant; and / or, the additive is glycerol and vitamin; preferably, in the Isochrysis preservation solution, for every 10 billion Isochrysis cells, the addition amount of glycerol is 50-500 mg, preferably 50-350 mg; Preferably, the vitamins include vitamin B 1 , vitamin B 12 and biotin; More preferably, based on the volume of the chrysophyta preservation solution, the concentration of the vitamin B 1 is 20-200 μg / L, preferably 50-150 μg / L; More preferably, based on the volume of the chrysophyte preservation solution, the concentration of the vitamin B 12 is 1-20 μg / L, preferably 2-10 μg / L; more preferably, based on the volume of the Isochrysis preservation solution, the concentration of biotin is 1-20 μg / L, preferably 2-10 μg / L.

10. A method for aquaculture, characterized in that, the method comprises: (1) Preserving the chrysophyta according to the method described in any one of claims 7-9, and thawing the cryopreservation; (2) Feeding the thawed product obtained in step (1) to aquaculture animals.

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