CN111394254A - Preparation method and application of isopulegolic algae powder - Google Patents
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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 isopulegon culture solution, step 2) performing high-density culture, and step 3) preparing algae powder. The algae powder is prepared 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 isopulegolic algae 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 larvae of the 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 culture medium composition disclosed by CN 106591137A comprises KNO 3: 300400 mg/L, KH2PO 4:10 mg/L, FeSO4.7H2O: 2.5 mg/L, Na2 EDTA: 10 mg/L, MnSO 4: 0.25 g/L: VB 1: 6 mg/L, VB 12: 50 ug/L, an additive: 0.53 g/L, a plant growth regulator prepared from 6-BA and naphthylacetic acid, wherein when the heteroglea is cultured, the heteroglea has the characteristics of high growth rate and high cell density, and the heteroglea is cultured by using a leather factory, so that the problems of repeated utilization of useful substances in the leather factory, improvement of heteroglea growth speed, improvement of growth speed, low growth rate of heteroglea component, low pollution of environmental pollution, low cost, low pollution of environmental pollution, low pollution cost, low pollution to water and the like are solved.
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 isopulegolic algae 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;
and 3) preparing algae powder, namely adding a flocculating agent into the algae liquid, wherein the adding amount is 10-20 mg: 1L, uniformly mixing, standing for 12-18h, discharging the liquid, collecting precipitates, then placing at the low temperature of-18 ℃, freezing for 30-90min, then placing at the temperature of 50-70 ℃ for drying to constant weight, and finally crushing into algae powder.
Preferably, the concentration of the ammonium bicarbonate is 30-50 mg/L.
Preferably, the concentration of the brassinolide is 0.02-0.03 mg/L.
Preferably, the seeding density is 200-.
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: 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 of 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 heteroglea under the red light condition, maintain the rapid proliferation of the heteroglea 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: the effect 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 to achieve the desired results with reference to 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 isocollophyte culture solution, namely improving an f/2 culture medium, namely adding ammonium bicarbonate and brassinolide into the f/2 culture medium, and controlling the concentrations of the ammonium bicarbonate and the brassinolide to be 50 mg/L and 0.03 mg/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)2S), the light-dark time ratio is 12:12, the air flow is introduced to be 0.5vvm, the algae liquid is obtained after the algae liquid is cultured for 9d, and sodium chloride aqueous solution with the concentration of 2 mol/L is fed in during the whole culture processThe salinity is 23;
and (2) preparing algae powder, namely adding a flocculating agent (prepared by uniformly mixing chitosan and zeolite powder according to the mass ratio of 1: 1) into the algae liquid, adding 10 mg: 1L, uniformly mixing, standing for 18h, discharging liquid, collecting precipitate, then placing at the low temperature of 18 ℃ below zero, freezing for 60min, then placing at the temperature of 60 ℃ for drying to constant weight, and finally crushing into algae powder.
Example 2
The preparation method of the isopulegolic algae powder comprises the following steps:
preparing an isocollophyte culture solution, namely improving an f/2 culture medium, namely adding ammonium bicarbonate and brassinolide into the f/2 culture medium, and controlling the concentrations of the ammonium bicarbonate and the brassinolide to be 30 mg/L and 0.02 mg/L respectively;
high-density culture: selecting vigorous and pollution-free Isoglena, inoculating into culture 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 of 740nm wavelength, and light intensity of 55 μmol/(m)2S), the light-dark time ratio is 14:10, the air flow is introduced to be 0.6vvm, and the culture is carried out for 8d, wherein the salinity is controlled to be 25 by feeding sodium chloride aqueous solution with the concentration of 3 mol/L in the whole culture process;
and (2) preparing algae powder, namely adding a flocculating agent into the algae liquid, uniformly mixing the flocculating agent and the algae liquid according to the adding amount of 20 mg: 1L, standing for 12 hours, discharging the liquid, collecting precipitates, then placing the precipitates at the low temperature of 18 ℃ below zero, freezing for 80min, then placing the precipitates at the temperature of 65 ℃ for drying to constant weight, and finally crushing the precipitates into the algae powder.
Example 3
The preparation method of the isopulegolic algae powder comprises the following steps:
preparing an isocollaronium culture solution, namely improving an f/2 culture medium, wherein ammonium bicarbonate and brassinolide are added into the f/2 culture medium, and the concentrations of the ammonium bicarbonate and the brassinolide are respectively controlled to be 40 mg/L and 0.025 mg/L;
high-density culture: selecting vigorous and pollution-free heteroglena, inoculating to an annular raceway pond containing a heteroglena culture solution, controlling the inoculation density to be 500 ten thousand/ml, and controlling the temperatureAt 25 deg.C, red light irradiation, electromagnetic radiation with wavelength of 730nm, and light intensity of 50 μmol/(m)2S), the light-dark time ratio is 10:14, the air flow is introduced to be 0.5vvm, and the culture is carried out for 7d, wherein the salinity is controlled to be 23 by feeding a sodium chloride aqueous solution with the concentration of 2 mol/L in the whole culture process;
and (2) preparing algae powder, namely adding a flocculating agent into the algae liquid, uniformly mixing the flocculating agent and the algae liquid according to the adding amount of 15 mg: 1L, standing for 16h, discharging the liquid, collecting precipitates, then placing the precipitates at the low temperature of 18 ℃ below zero, freezing for 90min, then placing the precipitates at the temperature of 60 ℃ for drying to constant weight, and finally crushing the precipitates into the algae powder.
Comparative example 1
A production process for culturing alloglena comprises the following steps:
high density culture, selecting vigorous and pollution-free Heterococcus and inoculating to culture pond containing f/2 culture medium, inoculating density is 500 ten thousand per ml, controlling temperature at 25 deg.C, irradiating with white light L ED (wavelength 450 and 465 nm), light intensity is 45 μmol/(m-2S), the light-dark time ratio is 12:12, the air flow is 0.5vvm, the culture is carried out for 9d, and the salinity is controlled to be 23 by feeding sodium chloride aqueous solution with the concentration of 2 mol/L in the whole culture process.
Example 4
1. firstly, adding ammonium bicarbonate, 0,10,20,30,40,50,60 and 70, and a unit of mg/L, as shown in figure 1, on the basis of the f/2 culture medium, wherein the biomass of the isocollagen is rapidly increased along with the increase of the culture time, the biomass is close to the maximum value after 9d, the culture time is continuously increased, the biomass is also slightly increased, but 9d is selected as the best in consideration of the production period, and the increase of the ammonium bicarbonate concentration can bring the increase of the algae density by longitudinally observing, taking the culture 9d as an example, the addition amount of the ammonium bicarbonate concentration of 50 mg/L leads the algae biomass to reach the maximum value, the ammonium bicarbonate concentration is continuously increased, and the ammonium bicarbonate concentration has no obvious influence on the algae biomass, so the culture 9d is selected, the ammonium bicarbonate concentration is 30-50 mg/L is the best, and particularly the ammonium bicarbonate concentration of 50 mg/L is selected.
2. The method comprises the steps of culturing for 9d, verifying the influence of brassinolide, sodium naphthaleneacetate and indolebutyric acid on the biomass of the heteroglea under the test condition that the ammonium bicarbonate addition concentration is 50 mg/L, setting the concentrations to be 0.01,0.02,0.03,0.04,0.05 and 0.06 respectively, wherein the unit is mg/L, as shown in figure 2, the indolebutyric acid with different concentrations hardly influences the biomass of the heteroglea, the small-amplitude increase of the biomass can be realized after the sodium naphthaleneacetate reaches 0.05 mg/L, the increase of the biomass can be realized by only within 5% compared with a non-added group, the use value is low, the brassinolide with low concentration has an obvious promoting effect on the biomass of the heteroglea, the optimum effect is realized when the addition amount of 0.03 mg/L is realized, the concentration is continuously increased, and the biomass of the heteroglea is not obviously.
3. The effect of different light sources on the biomass of alloglena.
In the culture period of 9d, the test conditions that the ammonium bicarbonate addition concentration is 50 mg/L and the brassinolide addition concentration is 0.03 mg/L are selected, and the test is carried out by selecting light with different wavelengths, wherein the white light L ED wavelength is 460nm, and the red light 640nm, 660nm, 680nm, 700nm, 720nm, 740nm and 760nm, as shown in figure 3, compared with the white light, the red light can improve the density of the heteroglena, the influence difference of the red light with different wavelengths on the heteroglena is large, the red light with the wavelength of 720 plus 740nm can greatly improve the density of the heteroglena, and compared with the white light, the density can be improved by about 40%.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but 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 (10)
1. 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.
2. The method of claim 1, comprising the steps of:
step 1) preparation of an isopulegol culture solution: adding ammonium bicarbonate and brassinolide into the f/2 culture medium to obtain an isopulegolic algae 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 flocculant into the algae liquid, mixing, standing for 12-18h, discharging the 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.
3. The method of claim 2, wherein the ammonium bicarbonate is present in a concentration of 30-50 mg/L.
4. The method of claim 2, wherein the brassinolide concentration is 0.02 to 0.03 mg/L.
5. The method as set forth in claim 2, wherein the seeding density is 200-1000 ten thousand/ml.
6. The method as claimed in claim 2, wherein the wavelength of red light is between 720 and 740 nm.
7. The method according to claim 2, wherein the intensity of red light is 45 to 55 μmol/(m)2.S）。
8. The preparation method of claim 2, wherein the flocculant is prepared by uniformly mixing chitosan and zeolite powder according to a mass ratio of 1: 1.
9. Isoalgae powder prepared by the method according to any one of claims 1 to 8.
10. Use of the isopulego flour of claim 9 in aquaculture.
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2020
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| US20040175407A1 (en) * | 2002-09-09 | 2004-09-09 | Reactive Surfaces, Ltd. | Microorganism coating components, coatings, and coated surfaces |
| US20170349892A1 (en) * | 2016-06-02 | 2017-12-07 | Reliance Industries Limited | Propiconazole resistant mutants of Chlorella Species |
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