CN110713933A

CN110713933A - Method for preserving microalgae bait

Info

Publication number: CN110713933A
Application number: CN201910932818.XA
Authority: CN
Inventors: 杨欢; 刘建国; 刘士涛; 吕小鸿; 姚然; 吴聪萍; 邹志刚
Original assignee: Shenzhen Huada Sansheng Garden Technology Co Ltd; Kunshan Innovation Institute of Nanjing University
Current assignee: Shenzhen Huada Sansheng Garden Technology Co Ltd; Kunshan Innovation Institute of Nanjing University
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2020-01-21

Abstract

The invention discloses a method for preserving microalgae bait, which comprises the steps of uniformly mixing a PH buffer solution, a cell protective agent, concentrated organic acid, an antiseptic and an antibiotic solution with the fermented microalgae bait with high cell concentration according to a certain volume ratio under the aseptic condition, and then putting the mixture into the condition of 0 ~ 10 ℃ and shading for storage.

Description

Method for preserving microalgae bait

Technical Field

The invention belongs to the technical field of aquatic living organism bait preservation, and particularly relates to a method for preserving microalgae bait.

Background

Microalgae is rich in protein, various essential amino acids, polyunsaturated fatty acids, vitamins, minerals, sterols and other bioactive substances, and because of the characteristics, microalgae cells are used as a common living body bait in aquaculture, and meanwhile, the microalgae bait can also play a role in purifying water. However, when the microalgae is used as bait, the number of living microalgae and the feeding time are closely matched with the seedling culture, no error exists, and otherwise, the bait supply and demand are easy to be disconnected. Currently, the main problem faced after large-scale cultivation of microalgae is how to ensure the number and activity of living cells of microalgae in the processes of high-temperature, long-distance transportation and storage. So that the bait can be used immediately when the fresh algae liquid is deficient, thereby thoroughly solving the problem of influence of unstable properties of the bait microalgae product on seedling production.

In order to solve the bottleneck problems of unstable characters, unbalanced nutritional components, untimely supply and the like commonly existing in the process of preservation and transportation of microalgae biological bait and reduce the adverse effects of the adverse factors on aquatic seedling production, the microalgae bait is mainly preserved by using a low-temperature drying technology at present, and the technology mainly has the following specific problems: (1) the activity of microalgae cells is completely lost, the palatability is reduced, and the dispersibility and shape retention of the microalgae in a water body are influenced. (2) The freezing preservation needs to add a high dose of cell antifreeze agent, and the water body can be polluted when the feed is thrown. (3) The operation process is complicated, the equipment requirement is high and the preservation cost is overhigh. As for the normal temperature preservation rule, the problems of vigorous metabolism, easy putrefaction, unsuitability for long-term preservation and the like of the algae exist. In addition, the preservation period of the existing cryopreservation technology is only 1-2 months generally, and the phenomenon of cell activity reduction and even death may occur in the later period of preservation, thus the service cycle and the transportation radius of the microalgae bait are seriously influenced.

Disclosure of Invention

In order to solve the problems, the invention provides a method for preserving microalgae bait.

The technical purpose is achieved, the technical effect is achieved, and the invention is realized through the following technical scheme:

a method for preserving microalgae bait comprises the steps of uniformly mixing a pH buffer solution, a cell protective agent, a concentrated organic acid, a preservative and an antibiotic solution with the microalgae bait according to a certain volume ratio under an aseptic condition, and then storing the mixture in a shading condition at the temperature of 0-10 ℃.

As a further improvement of the invention, the PH buffer solution is a PBS buffer system or a Tris-HCl buffer system, the PH of the biological culture solution is controlled to be 6.2-7.2, and the PH buffer solution is added into the microalgae bait according to the proportion of 1-2% of the volume ratio.

As a further improvement of the invention, the organic acid is selected from any one of citric acid or ascorbic acid, and the organic acid is added into the microalgae bait according to the proportion of 0.1-0.2 percent by volume.

As a further improvement of the invention, the preservative is selected from any one or more of potassium sorbate, sodium benzoate and calcium propionate, and the preservative is added into the microalgae bait according to the volume ratio of 0.05-0.2%.

As a further improvement of the invention, the antibiotic is selected from any one or more of penicillin, chloramphenicol or cefamycin, and the preservative is added into the microalgae bait according to the volume ratio of 0.01-0.03%.

As a further improvement of the invention, the cell protective agent is selected from glycerol, and the glycerol is added into the microalgae bait according to the proportion of 1-5% by volume.

As a further improvement of the invention, the number of living cells in the microalgae bait is more than 20 hundred million/ml, and the dry weight of the algae cells in the microalgae bait reaches 50-120 g/L.

As a further improvement of the invention, the microalgae bait is selected from sterile diatom, chrysophyceae or chlorella bait.

As a further improvement of the invention, the method comprises the step of placing the sterilized PH buffer solution, the cell protective agent, the concentrated organic acid, the preservative and the antibiotic in the sterilized instrument to be mixed with the microalgae bait in a sterile environment.

The invention has the beneficial effects that:

(1) the method is simple to operate, the added substances do not produce toxic action on microalgae cells, but maintain the acid-base system of the microalgae bait and avoid the pollution of mixed bacteria during preservation;

(2) the method can keep the survival rate and the biological activity of the microalgae cells to the maximum extent, can prolong the preservation period of the existing bait by 1-2 months, and has good application prospect;

(3) the method mainly aims at the preservation of the microalgae bait with high cell concentration, the added amount of the antiseptic and the antibiotic is lower, and the investment amount of the method is less than that of the bait algae with low cell concentration in the culture water body with the same volume. Therefore, the water body is not polluted by N, P and other nutrients brought into the water body by the microalgae bait and a small amount of antiseptic, antibiotic and the like in the bait preservation system.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Experimental group 1: firstly, respectively preparing 1g/L citric acid, 100g/L potassium sorbate and 100g/L penicillin mother liquor and 10 times of PBS buffer solution with pH of 6.8, placing the potassium sorbate solution and glycerol into a high-temperature sterilization pot, sterilizing at 121 ℃ for 20min, and filtering the penicillin and citric acid solutions by using a 0.22 mu m sterile filter for later use. 5mL of sterile PBS buffer solution, 0.5mL of citric acid solution, 0.15mL of potassium sorbate, 0.5mL of penicillin and 25mL of glycerol are added into bait algae in a sterile room, mixed uniformly, sealed and stored in a refrigerator at 4 ℃.

Experimental group 2: firstly, respectively preparing 1g/L citric acid, 100g/L potassium sorbate and 100g/L penicillin mother liquor and 10 times of PBS buffer solution with pH of 6.8, placing the potassium sorbate solution and glycerol into a high-temperature sterilization pot, sterilizing at 121 ℃ for 20min, and filtering the penicillin and citric acid solutions by using a 0.22 mu m sterile filter for later use. Sterile PBS buffer solution 10mL, citric acid solution 0.25mL, potassium sorbate 0.15mL, penicillin 0.5mL and glycerin 20mL are added into bait algae in a sterile room, mixed uniformly, sealed and stored in a refrigerator at 4 ℃.

Experimental group 3: firstly, respectively preparing 1g/L citric acid, 100g/L potassium sorbate and 100g/L penicillin mother liquor and 10 times of PBS buffer solution with pH of 6.8, placing the potassium sorbate solution and glycerol into a high-temperature sterilization pot, sterilizing at 121 ℃ for 20min, and filtering the penicillin and citric acid solutions by using a 0.22 mu m sterile filter for later use. 5mL of sterile PBS buffer solution, 1.0mL of citric acid solution, 0.05mL of potassium sorbate, 0.5mL of penicillin and 25mL of glycerol are added into bait algae in a sterile room, mixed uniformly, sealed and stored in a refrigerator at 4 ℃.

Blank group: and (3) directly placing the filled microalgae bait into a refrigerator at 4 ℃ for preservation without adding any chemical substance.

Each experimental group was subjected to 3 parallel experiments, and samples were taken when the samples were preserved for 30d, 60d, and 90d, respectively, and the activity of algal cells was measured by flow cytometry, and the total number of colonies was measured by plate coating.

The results of the four groups are shown in the following table:

TABLE 1 cell Activity and colony count for Chlorella pyrenoidosa bait Collection

As can be seen from the table, the cell activity and survival rate of the microalgae baits in the experimental groups 1 to 3 are much higher than those in the blank group after the microalgae baits are preserved for 90 days. And with the prolonging of preservation time, the activity of the bait cells of the blank group is reduced at a higher speed, and the total number of colonies is increased sharply. The results prove that the long-term microalgae bait preservation method provided by the invention can effectively maintain the biological activity and the survival rate of the bait algae cells and inhibit the growth of microorganisms.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method for preserving microalgae bait is characterized in that under the aseptic condition, a pH buffer solution, a cell protective agent, a concentrated organic acid, a preservative and an antibiotic solution are uniformly mixed with the microalgae bait according to a certain volume ratio, and then the mixture is placed at the temperature of 0 ~ 10 ℃ and stored in a shading condition.

2. A method of preserving microalgal bait according to claim 1, wherein: the PH buffer solution is a PBS buffer system or a Tris-HCl buffer system, the PH of the biological culture solution is controlled to be 6.2-7.2, and the PH buffer solution is added into the microalgae bait according to the proportion of 1-2% of the volume ratio.

3. A method of preserving microalgal bait according to claim 1, wherein: the organic acid is selected from any one of citric acid or ascorbic acid, and is added into the microalgae bait according to the volume ratio of 0.1-0.2%.

4. A method of preserving microalgal bait according to claim 1, wherein: the preservative is selected from one or more of potassium sorbate, sodium benzoate and calcium propionate, and is added into the microalgae bait according to the volume ratio of 0.05-0.2%.

5. A method of preserving microalgal bait according to claim 1, wherein: the antibiotic is selected from any one or more of penicillin, chloramphenicol and cefamycin, and the preservative is added into the microalgae bait according to the volume ratio of 0.01-0.03%.

6. A method of preserving microalgal bait according to claim 1, wherein: the cell protective agent is selected from glycerol, and the glycerol is added into the microalgae bait according to the proportion of 1-5% by volume.

7. The method for preserving microalgae bait according to claim 1, wherein the number of living cells in the microalgae bait is more than 20 hundred million/ml, and the dry weight of the algae cells in the microalgae bait reaches 50 ~ 120 g/L.

8. A method of preserving microalgal bait according to claim 1 or 7, characterized in that: the microalgae bait is selected from sterile diatom, chrysophyceae or green algae bait.

9. A method of preserving microalgal bait according to claim 1, wherein: comprises placing sterilized PH buffer solution, cell protectant, concentrated organic acid, antiseptic and antibiotic in a sterilized instrument, and mixing with microalgae bait.