CN111394254B - Preparation method and application of isopulva powder - Google Patents
Preparation method and application of isopulva powder Download PDFInfo
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- CN111394254B CN111394254B CN202010315157.9A CN202010315157A CN111394254B CN 111394254 B CN111394254 B CN 111394254B CN 202010315157 A CN202010315157 A CN 202010315157A CN 111394254 B CN111394254 B CN 111394254B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/12—Unicellular algae; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/02—Separating microorganisms from their culture media
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention belongs to the technical field of algae, and discloses a preparation method of isopulegolic algae powder, which comprises the following steps: step 1) preparing an isopulegolic algae culture solution, step 2) performing high-density culture, and step 3) preparing algae powder. The invention prepares the algae powder by flocculating and precipitating algae cells and then freeze-drying and crushing, can be applied to aquaculture and has extremely high market application value.
Description
Technical Field
The invention belongs to the technical field of algae, and particularly relates to a preparation method and application of heteroglena powder.
Background
The microalgae is rich in nutrient substances, and many algae contain high protein, and also contain vitamins and trace elements which are necessary for maintaining the growth of aquatic product seedlings, so that the microalgae has an obvious promotion effect on various physiological functions of the seedlings. For example, the vitamin E has the effects of promoting phagocytosis of macrophages of aquatic animals, improving complement activity and enhancing the immunity of young aquatic animals; the addition of anabaena powder in the compound feed for prawns can obviously improve the growth speed and disease resistance of prawns. Therefore, the excellent algae species such as anabaena, chlorella and the like have good application prospect in the cultivation of fish and shrimp fries. The unicellular algae in a certain proportion is added into the compound feed to serve as bait in the early stage of fish and shrimp seedling raising, the survival rate of seedlings is higher, and the seedlings can be raised with high and stable yield. Therefore, in many areas, the larvae are cultivated by adding non-biological baits to unicellular algae. Different algae species have certain differences in the content of nutritional indexes. The mixed regulation and control mode of various algae is beneficial to the balanced supply of various nutrient elements, thereby ensuring the growth, development and smooth metamorphosis of the larva. Algae species are complicated and the amount is large, but only a few kinds of bait algae which are widely applied in aquaculture at present are provided, wherein chlorella (the first artificially cultured microalgae in China), dunaliella salina, chaetoceros, phaeodactylum tricornutum, equilateral chrysophyceae and the like exist, and in addition, skeletonema costatum, heteroglena, tetraselmis, nitzschia closterium also gradually start to be cultured.
Besides being used as direct bait, the microalgae has another important function of being used for feeding secondary baits such as rotifers, artemia, copepods, cladocerans and the like, and can obviously enhance the content of polyunsaturated fatty acids and various vitamins contained in organisms of the secondary baits so as to meet the requirement of aquatic animal larvae on high-quality secondary baits. The wide application of the excellent algae species powerfully promotes the development of the seedling industry and also promotes the progress of the seedling raising technology. With the increasing attention paid to the concept of healthy cultivation, it is expected that the algae will certainly play more and more important roles in the offspring seed cultivation of aquatic economic animals in the future.
The heteroglea is good bait algae, but the research on artificial high-density culture is less. Only a few reports exist at present, and CN 106591137A discloses an isoglena culture medium composition, which comprises the following components: KNO3:300 400mg/L; KH2PO4:10mg/L; feso4.7h2o:2.5mg/L; na2EDTA:10mg/L; mnSO4:0.25g/L: VB1:6mg/L; VB12:50ug/L; additive: 0.5 3g/L; the plant growth regulator prepared from 6-BA and naphthylacetic acid has the advantages of high growth rate and high cell density when culturing the heteroglea. CN 104130945A utilizes leather factory waste water to cultivate heteroglena, can realize the reuse of useful substances in the leather factory waste water and can improve the growth speed and the nutrient content of the heteroglena, thereby achieving the double effects of protecting the environment and producing biological resources, having simple, convenient and quick operation, low cost, no pollution, high social, economic and ecological benefits, solving the problem of waste water pollution of the leather factory, solving the defects of slow growth, low yield, poor benefits, easy pollution and low nutrient content of the heteroglena, and realizing the production culture of the heteroglena. However, the method has the defects of high cost, unclean water body, easy carrying of pollutants, low density, poor environmental friendliness and the like.
The culture conditions of different algae are different greatly, and the algae can not be used and carried. The previous patent technology of the applicant, namely 'a production process for culturing the heteroglea at high density', greatly improves the biomass of the heteroglea by optimizing a culture medium and culture conditions. On the basis, the applicant continues to collect and process the heteroglena to prepare products that can be used in the aquaculture industry.
Disclosure of Invention
In order to solve the technical defects in the prior art, the invention provides a preparation method and application of isopulegolic algae powder.
The invention is realized by the following technical scheme:
the preparation method of the isopulegolic algae powder comprises the following steps: step 1) preparing an isopulegon culture solution, step 2) performing high-density culture, and step 3) preparing algae powder.
Further, the preparation method comprises the following steps:
step 1) preparation of an isopulegol culture solution: adding ammonium bicarbonate and brassinolide into the f/2 culture medium to obtain an isopachromyces culture solution;
step 2) high-density culture: selecting vigorous and pollution-free heteroglena, inoculating to a culture pond containing a heteroglena culture solution, controlling the temperature at 23-28 ℃, irradiating with red light, introducing air flow of 0.4-0.6vvm, and culturing for 7-9d to obtain an algae solution; in the whole culture process, the salinity is controlled to be 22-28 by feeding a sodium chloride aqueous solution;
step 3), preparing algae powder: adding a flocculating agent into the algae liquid, wherein the adding amount is 10-20mg:1L, mixing, standing for 12-18h, discharging liquid, collecting precipitate, freezing at-18 deg.C for 30-90min, drying at 50-70 deg.C to constant weight, and pulverizing into algae powder.
Preferably, the concentration of the ammonium bicarbonate is 30-50mg/L.
Preferably, the concentration of the brassinolide is 0.02-0.03mg/L.
Preferably, the inoculation density is 200-1000 ten thousand per ml.
Preferably, the wavelength of the red light is between 720-740 nm.
Preferably, the light intensity of the red light is 45-55 [ mu ] mol/(m) 2 .S）。
Preferably, the flocculant is prepared by uniformly mixing chitosan and zeolite powder according to the mass ratio of 1.
On the other hand, the invention also claims the isopulegolic algae powder prepared by the preparation method and the application thereof in aquaculture.
The beneficial effects obtained by the invention mainly comprise the following aspects:
the near infrared light with a certain wavelength can more effectively stimulate a photoreaction system of the heteroglene and stimulate the heteroglene to be divided and proliferated more quickly, thereby realizing high-density culture in a short time.
After inoculation, a short delay period appears, the culture reaches a higher increase for 3d until 7-9d, then a stationary period is entered, and the increase rate is obviously reduced, so that the control of the culture period to be 7-9 days is more suitable.
The f/2 culture medium is suitable for most algae to grow, but is not the optimal culture medium for most algae, and on the basis of the f/2 culture medium, the ammonium bicarbonate is added, so that the absorption of nitrogen by the alloglena can be promoted, carbon dioxide can be provided for the alloglena to utilize, the growth of algae cells is stimulated, the biomass is improved, and no environmental pollution substances are generated; the brassinolide can obviously promote the cell division of the heteroglena under the red light condition, maintain the rapid proliferation of the heteroglena and improve the growth amount of the algae cells.
The algae powder is prepared by flocculating and precipitating algae cells and then freeze-drying and crushing, can be independently applied or combined with other algae in aquaculture, and has extremely high market application value.
The flocculant used in the invention is prepared from chitosan and zeolite powder, and not only has a flocculation function, but also has other functions; wherein the chitosan can stimulate immune system and improve immunity, and is also a growth promoter and a bacteriostatic agent; the zeolite powder can provide minerals for aquaculture, and is helpful for the proliferation of aquatic animals.
Drawings
FIG. 1: the effect of ammonium bicarbonate on the biomass of heteroglena;
FIG. 2 is a schematic diagram: the influence of brassinolide on the biomass of heteroglena;
FIG. 3: influence of different wavelengths of light on biomass of the heteroglena.
Detailed Description
Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
The preparation method of the isopulegolic algae powder comprises the following steps:
preparing an isopulegolic algae culture solution: the f/2 culture medium is improved, and the method comprises the following steps: adding ammonium bicarbonate and brassinolide into the f/2 culture medium, and controlling the concentrations of the ammonium bicarbonate and the brassinolide to be 50mg/L and 0.03mg/L respectively;
high-density culture: selecting vigorous and pollution-free Isoglena, inoculating into culture pond containing Isoglena culture solution, inoculating at density of 500 ten thousand per ml, controlling temperature at 25 deg.C, irradiating with red light with electromagnetic radiation of 720nm wavelength, and light intensity of 45 μmol/(m) 2 S), the light-dark time ratio is 12, the air flow is 0.5vvm, and the algae solution is obtained after 9d culture; in the whole culture process, the salinity is controlled to be 23 by feeding a sodium chloride aqueous solution with the concentration of 2 mol/L;
preparing algae powder: adding a flocculating agent (prepared by uniformly mixing chitosan and zeolite powder according to the mass ratio of 1: 1L, mixing, standing for 18h, discharging liquid, collecting precipitate, freezing at-18 deg.C for 60min, drying at 60 deg.C to constant weight, and pulverizing into algae powder.
Example 2
The preparation method of the isopulegolic algae powder comprises the following steps:
preparing an isopulegolic algae culture solution: the f/2 culture medium is improved, and the specific steps are as follows: adding ammonium bicarbonate and brassinolide into the f/2 culture medium, and controlling the concentrations of the ammonium bicarbonate and the brassinolide to be 30mg/L and 0.02mg/L respectively;
high-density culture: selecting the materials with vigorous growthInoculating non-pollution Isogloea into culture pond containing Isogloea culture solution at an inoculation density of 500 ten thousand per ml, at 25 deg.C, under irradiation of red light with 740nm wavelength electromagnetic radiation, and with light intensity of 55 μmol/(m) 2 S), the light-dark time ratio is 14, the air flow is 0.6vvm, and the culture is carried out for 8d; in the whole culture process, the salinity is controlled to be 25 by feeding 3mol/L sodium chloride aqueous solution;
preparing algae powder: adding a flocculating agent into the algae liquid, wherein the adding amount is 20mg:1L, mixing, standing for 12h, discharging liquid, collecting precipitate, freezing at-18 deg.C for 80min, drying at 65 deg.C to constant weight, and pulverizing into algae powder.
Example 3
The preparation method of the isopulegolic algae powder comprises the following steps:
preparing an isopulegolic algae culture solution: the f/2 culture medium is improved, and the specific steps are as follows: adding ammonium bicarbonate and brassinolide into the f/2 culture medium, wherein the concentrations of the ammonium bicarbonate and the brassinolide are controlled to be 40mg/L and 0.025mg/L respectively;
high-density culture: selecting vigorous and pollution-free Isoglena, inoculating into annular raceway pond containing Isoglena culture solution at an inoculation density of 500 ten thousand per ml, controlling temperature at 25 deg.C, irradiating with red light having electromagnetic radiation with wavelength of 730nm, and light intensity of 50 μmol/(m) under irradiation of red light 2 S), the light-dark time ratio is 10, the air flow is 0.5vvm, and the culture is carried out for 7d; in the whole culture process, the salinity is controlled to be 23 by feeding a sodium chloride aqueous solution with the concentration of 2 mol/L;
preparing algae powder: adding a flocculating agent into the algae liquid, wherein the adding amount is 15mg:1L, mixing, standing for 16h, discharging liquid, collecting precipitate, freezing at-18 deg.C for 90min, drying at 60 deg.C to constant weight, and pulverizing into algae powder.
Comparative example 1
A production process for culturing heteroglena comprises the following steps:
high-density culture: selecting vigorous and pollution-free heteroglena to be inoculatedInoculating into culture pond containing f/2 culture medium at 25 deg.C with density of 500 ten thousand per ml and light intensity of 45 μmol/(m) under irradiation of white light LED (wavelength of 450-465 nm) 2 S), the light-dark time ratio is 12, the air flow rate is 0.5vvm, and the culture is carried out for 9d; the salinity is controlled to be 23 by feeding sodium chloride aqueous solution with the concentration of 2mol/L in the whole culture process.
Example 4
1. The f/2 culture medium is an algae conventional culture medium, the invention is improved on the basis of the conventional culture mode of the comparative example 1, and the influence of ammonium bicarbonate and brassinolide on the biomass of the isocollagen is verified. Firstly, adding ammonium bicarbonate 0,10,20,30,40,50,60 and 70 in mg/L on the basis of f/2 culture medium, as shown in figure 1, when the culture time is increased, the biomass of the heteroglea is rapidly increased, about 9d is close to the maximum value, the culture time is continuously increased, the biomass is slightly increased, but the 9d is selected as the best considering the production period; longitudinally observing that the density of the algae is increased due to the increase of the ammonium bicarbonate concentration, taking the culture time of 9d as an example, the addition amount of 50mg/L of the ammonium bicarbonate concentration enables the biomass of the algae to reach the maximum value, and the ammonium bicarbonate concentration is continuously increased without obvious influence on the biomass of the algae, so the culture time of 9d is selected, and the ammonium bicarbonate concentration is optimally 30-50mg/L, particularly 50mg/L.
2. Verifying the influence of brassinolide, sodium naphthaleneacetate and indolebutyric acid on the biomass of the isocollagen under the test condition that the ammonium bicarbonate addition concentration is 50mg/L after the culture is carried out for 9 days; the concentrations are respectively set to be 0.01,0.02,0.03,0.04,0.05 and 0.06, the unit is mg/L, as shown in figure 2, indolebutyric acid with different concentrations hardly influences the biomass of the heteroglena, and after the sodium naphthaleneacetate reaches 0.05mg/L, the biomass is improved to a small extent, and compared with a group without the indolebutyric acid, the concentration can be improved by only within 5 percent, and the use value is low; the brassinolide with low concentration has obvious promotion effect on the biomass of the heteroglena, the best addition amount of the brassinolide is reached when the addition amount is 0.03mg/L, the concentration is continuously increased, and the biomass of the heteroglena is not obviously improved.
3. The effect of different light sources on the biomass of alloglena.
Selecting light with different wavelengths for testing under the test conditions that the ammonium bicarbonate addition concentration is 50mg/L and the brassinolide addition concentration is 0.03mg/L after the culture is carried out for 9d, wherein the wavelength of a white light LED is 460nm; red light 640nm, 660nm, 680nm, 700nm, 720nm, 740nm and 760nm; as shown in FIG. 3, compared with white light, red light can increase the density of the heteroglea, the influence difference of the red light with different wavelengths on the heteroglea is larger, and the density of the heteroglea can be greatly increased by the red light with the wavelength of 720-740nm, which can be increased by about 40% compared with the white light.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (2)
1. The preparation method of the isopulegolic algae powder comprises the following steps:
step 1) preparing an isopachal culture solution: adding ammonium bicarbonate and brassinolide into the f/2 culture medium to obtain an isopulegolic algae culture solution;
the concentration of the ammonium bicarbonate is 30-50mg/L;
the concentration of the brassinolide is 0.02-0.03mg/L;
step 2) high-density culture: selecting vigorous and pollution-free heteroglena, inoculating to a culture pond containing heteroglena culture solution, controlling the temperature at 23-28 deg.C, irradiating with red light, introducing air flow of 0.4-0.6vvm, and culturing for 7-9d to obtain algae solution; in the whole culture process, the salinity is controlled to be 22-28 by feeding a sodium chloride aqueous solution;
the wavelength of the red light is 720-740 nm;
the light intensity of the red light is 45-55 mu mol/(m) 2 .S）；
Step 3), preparing algae powder: adding a flocculating agent into the algae liquid, mixing uniformly, standing for 12-18h, discharging the liquid, collecting the precipitate, then placing at a low temperature of-18 ℃, freezing for 30-90min, then placing at a temperature of 50-70 ℃, drying to constant weight, and finally crushing into algae powder;
the flocculant is prepared by uniformly mixing chitosan and zeolite powder according to the mass ratio of 1.
2. The method of claim 1, wherein the seeding density is 200 to 1000 ten thousand per ml.
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| CN106591137A (en) * | 2016-12-30 | 2017-04-26 | 宁波浮田生物技术有限公司 | Heterogloea sp. culture medium composition |
| CN108048330A (en) * | 2017-11-28 | 2018-05-18 | 东华大学 | Diatom soil matrix lotus positive electricity green flocculant is to the collecting method of selenium-rich chlorella product |
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| US10561115B2 (en) * | 2016-06-02 | 2020-02-18 | Reliance Industries Limited | Propiconazole resistant mutants of Chlorella species |
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| CN106591137A (en) * | 2016-12-30 | 2017-04-26 | 宁波浮田生物技术有限公司 | Heterogloea sp. culture medium composition |
| CN108048330A (en) * | 2017-11-28 | 2018-05-18 | 东华大学 | Diatom soil matrix lotus positive electricity green flocculant is to the collecting method of selenium-rich chlorella product |
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