Culture method for improving duckweed protein

Abstract

The invention relates to a culture method for improving duckweed protein, which comprises the following steps: purifying duckweed in a Hoagland working solution to obtain purified duckweed; mixing the biogas slurry with the Hoagland working solution to prepare a duckweed culture solution; wherein the concentration of the biogas slurry is 1.5%-5%, and the concentration of the Hoagland working solution is 95%-98.5%; after the purified duckweed is put into the duckweed culture solution, the duckweed culture solution is put into an incubator with preset culture conditions to be cultured, and the cultured duckweed is obtained. By adding the biogas slurry into the Hoagland working solution, the protein content of the cultured duckweed can be remarkably increased, the biogas slurry can be efficiently utilized, water resources are saved, an ideal way is provided for wastewater recycling, meanwhile, nitrogen and phosphorus in the biogas slurry can be utilized for promoting synthesis of duckweed protein, the protein synthesis efficiency is improved, and the yield of duckweed is increased. And a good basis is provided for feed research of duckweed.

Description

Technical Field

[0001] The invention relates to the technical field of duckweed cultivation, in particular to a cultivation method for improving duckweed protein. Background Art

[0002] Duckweed is a monocotyledonous aquatic floating plant belonging to the subfamily of the Araceae family. It is distributed in all kinds of freshwater bodies in my country due to its simple structure and strong adaptability. At the same time, duckweed is rich in starch, protein, vitamins and mineral elements, and is an important potential source of food, feed and bioenergy.

[0003] At present, the use of duckweed instead of soybean meal in feed is a popular research. It has the characteristics of fast growth, high protein content, low fiber content, easy harvesting and no serious pests, and has broad application prospects. Among them, there are two most commonly used technologies for cultivating duckweed, one is to use Hoagland nutrient solution for cultivation, and the other is to use Hunter nutrient solution for cultivation. Relatively speaking, the nitrogen-phosphorus ratio of the former is 6.5 times that of the latter, so Hoagland nutrient solution is more conducive to the growth of duckweed.

[0004] However, in order to stabilize the growth and protein content of duckweed, the Hoagland nutrient solution needs to be replaced every three days or so to ensure the stability of the nutrient content of the culture solution and the stable growth of duckweed. However, frequent replacement of the nutrient solution will not only increase the culture cost, but also affect the large-scale culture of duckweed. In addition, although the Hoagland nutrient solution has a certain positive effect on the growth rate and protein content of duckweed, it still cannot achieve a relatively ideal effect, that is, it cannot be used in the production of duckweed feed.

[0005] Therefore, providing a cultivation method that can reduce the use of nutrient solution, increase the protein content of duckweed, and realize feed production is an urgent problem to be solved. Summary of the invention

[0006] In view of this, the embodiments of the present application provide a method for culturing duckweed to increase duckweed protein to solve the above problems.

[0007] In a first aspect, the present invention provides a method for increasing duckweed protein, comprising the following steps: Purifying duckweed in Hoagland's working solution to obtain purified duckweed; Mixing the biogas slurry with the Hoagland working solution to prepare a duckweed culture solution; wherein the concentration of the biogas slurry is 1.5%-5%, and the concentration of the Hoagland working solution is 95%-98.5%; After the purified duckweed is added to the duckweed culture solution, it is placed in a culture box with preset culture conditions for culture to obtain cultured duckweed.

[0008] In some embodiments, the Hoagland working solution comprises the following components: Magnesium sulfate heptahydrate 49.2 μg / L, calcium nitrate tetrahydrate 108.6 μg / L, potassium dihydrogen phosphate 27.2 μg / L, potassium nitrate 50.2 μg / L, boric acid 0.286 μg / L, manganese chloride tetrahydrate 0.186 μg / L, zinc sulfate heptahydrate 0.022 μg / L, sodium molybdate dihydrate 0.009 μg / L, copper sulfate pentahydrate 0.009 μg / L, iron (II) sulfate heptahydrate 1.98 μg / L and disodium ethylenediaminetetraacetic acid 6 μg / L.

[0009] In some embodiments, the Hoagland working solution is obtained by adding pure water to the Hoagland reserve solution to make up the volume; wherein the Hoagland reserve solution comprises the following components: Magnesium sulfate heptahydrate 49.2 g / L, calcium nitrate tetrahydrate 108.6 g / L, potassium dihydrogen phosphate 27.2 g / L, potassium nitrate 50.2 g / L, boric acid 2.86 g / L, manganese chloride tetrahydrate 1.86 g / L, zinc sulfate heptahydrate 0.22 g / L, sodium molybdate dihydrate 0.09 g / L, copper sulfate pentahydrate 0.09 g / L, iron (II) sulfate heptahydrate 1.98 g / L and disodium ethylenediaminetetraacetic acid 6 g / L.

[0010] In some embodiments, the step of purifying duckweed in Hoagland's working solution to obtain purified duckweed includes: Obtaining duckweed and removing debris from the duckweed to obtain the pretreated duckweed; The pretreated duckweed was placed in a culture pot, and the Hoagland working solution was added for purification for 1 week to obtain purified duckweed.

[0011] In some embodiments, the duckweed species is D. sparsely veined or D. oligorrhiza.

[0012] In some of the embodiments, when the biogas slurry is mixed with the Hoagland working solution, the concentration of the Hoagland working solution is 95%-98%, and the concentration of the biogas slurry is 2%-5%.

[0013] In some of the embodiments, the total nitrogen content in the biogas slurry is 980 mg / L, and the total phosphorus content is 90 mg / L.

[0014] In some embodiments, the step of adding the purified duckweed to the duckweed culture solution and then placing the duckweed in a culture box with preset culture conditions for culture to obtain the cultured duckweed includes: Obtain the purified duckweed, remove impurities and absorb moisture from leaves and roots in sequence to obtain duckweed to be cultivated; Putting the duckweed to be cultured into a culture basin containing the culture solution; wherein the leaf surface of the duckweed to be cultured faces upward; The culture pot is placed in the culture box and cultured for 5 days according to the preset culture conditions to obtain cultured duckweed.

[0015] In some embodiments, when the duckweed to be cultured is put into a culture basin containing the culture solution, the duckweed covers more than 60% of the surface area of ​​the culture solution.

[0016] In some embodiments, the preset culture conditions include: The light duration is 16h, the dark duration is 8h, the light intensity is 6000LUX, the temperature is 25℃ and the humidity is 75%.

[0017] Technical effects of the present invention: By adding biogas slurry to the Hoagland working solution, the protein content of the cultured duckweed can be significantly increased, which can not only efficiently utilize the pig farm biogas slurry, save water resources, and provide an ideal way to recycle aquaculture wastewater and reduce sewage treatment costs, but also use the nitrogen and phosphorus in the biogas slurry to promote the synthesis of duckweed protein, enhance protein synthesis efficiency, reduce the use of nutrient solution, or even completely replace the nutrient solution in production practice to obtain a stable and reliable duckweed feed by breeding a large number of duckweed, providing a good foundation for the research on duckweed feed. In addition, since duckweed can grow all year round and has a wide range of adaptability to horizontal pH values, it can grow normally in the pH range of 5-9, can adapt to the ecological conditions of biogas slurry, does not require additional fertilization or irrigation, and does not compete with crop production for land.

[0018] Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the disclosure and, together with the description, serve to explain the principles of the disclosure.

[0020] Figure 1 A flow chart showing a method for cultivating duckweed protein in accordance with an embodiment of the present application is shown; Figure 2 The graph showing the results of measuring the protein content of Lemna sparsely veined is shown in the embodiment of the present application; Figure 3 Shown is a graph showing the results of measuring the protein content of Spirodela oligorrhiza in an example of the present application. DETAILED DESCRIPTION

[0021] Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0022] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0023] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present disclosure can also be implemented without certain specific details. In some examples, methods, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present disclosure.

[0024] Embodiment 1 The present application embodiment provides a method for increasing the culture of duckweed protein. Figure 1 is a flow chart of the steps of the method for cultivating duckweed protein according to an embodiment of the present application, such as Figure 1 As shown, the method comprises the following steps: S100, purifying duckweed in Hoagland's working solution to obtain purified duckweed; In this step, before formally culturing duckweed, duckweed needs to be purified in Hoagland's working solution, that is, there is a pre-purification process, the purpose of which is to reduce the impact of other impurities on the duckweed culture results, so as to obtain duckweed products with consistent growth status.

[0025] Therefore, before the purification process, it is necessary to prepare the Hoagland working solution in advance. Among them, the Hoagland working solution, as a nutritionally balanced culture medium, includes various mineral elements required for plant production. Specifically, it includes potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), molybdenum (Mo) and boron (B). After mixing with the biogas slurry, the above elements can synergize with the nitrogen and phosphorus in the biogas slurry to promote the growth of duckweed and protein synthesis.

[0026] In some of the embodiments, the Hoagland working solution includes the following ingredients: 49.2 μg / L magnesium sulfate heptahydrate, 108.6 μg / L calcium nitrate tetrahydrate, 27.2 μg / L potassium dihydrogen phosphate, 50.2 μg / L potassium nitrate, 0.286 μg / L boric acid, 0.186 μg / L manganese chloride tetrahydrate, 0.022 μg / L zinc sulfate heptahydrate, 0.009 μg / L sodium molybdate dihydrate, 0.009 μg / L copper sulfate pentahydrate, 1.98 μg / L iron (II) sulfate heptahydrate and 6 μg / L disodium ethylenediaminetetraacetic acid.

[0027] The Hoagland working solution in this embodiment is preferably prepared by mixing MgSO4.7H2O, Ca (NO3)2.4H2O, KH2PO4, KNO3, H3BO3, MnCL2.4H2O, ZnSO4.7H2O, Na2MoO4.2H2O, CuSO4.5H2O, FeSO4.7H2O, and Na2EDTA.

[0028] In some of the embodiments, the Hoagland working solution is obtained by adding pure water to the Hoagland stock solution to make up the volume; wherein the Hoagland stock solution comprises the following components: 49.2 g / L magnesium sulfate heptahydrate, 108.6 g / L calcium nitrate tetrahydrate, 27.2 g / L potassium dihydrogen phosphate, 50.2 g / L potassium nitrate, 2.86 g / L boric acid, 1.86 g / L manganese chloride tetrahydrate, 0.22 g / L zinc sulfate heptahydrate, 0.09 g / L sodium molybdate dihydrate, 0.09 g / L copper sulfate pentahydrate, 1.98 g / L iron (II) sulfate heptahydrate and 6 g / L disodium ethylenediaminetetraacetic acid.

[0029] Specifically, the preparation is carried out according to the components and dosages in Table 1. First, 11 500mL blue-capped glass reagent bottles are prepared, each reagent is prepared according to the concentration of the stock solution, and the required weight of nutrient salt powder is weighed and dissolved in pure water, and the volume is fixed and placed in the reagent bottle for standby use, so that each reagent can be stored separately to prevent some reagents from reacting for a long time. The stock solution in the reagent bottle is stored at room temperature for no more than 3 months.

[0030] Furthermore, when in use, the above reagent stock solutions are mixed according to the concentration and dosage of the working solution in Table 1, and then the volume is fixed with pure water to prepare the Hoagland working solution, which is used to mix with the biogas slurry in proportion when preparing duckweed culture solution to synergistically increase the protein content of duckweed.

[0031]

[0032] Table 1 Reference table for the preparation composition of Hoagland working solution In some of the embodiments, duckweed is purified in Hoagland's working solution to obtain purified duckweed, including: obtaining duckweed and removing debris on the duckweed to obtain pretreated duckweed; placing the pretreated duckweed in a culture basin, and adding Hoagland's working solution for purification for 1 week to obtain purified duckweed.

[0033] In the present embodiment, before formal cultivation, duckweed is cultivated with Hoagland working solution for 1 week, i.e. 7 days. Specifically, duckweed is fished out of the water with a scoop net having a preferred aperture of less than 1 mm, and debris on the leaves and roots, especially sundries, are rinsed with tap water to reduce the influence of other algae and impurities on duckweed cultivation. Subsequently, the moisture on the leaves and roots is dried with absorbent paper. Further, 2g of fresh duckweed is weighed and placed in a 2L culture pot, 1L of pre-prepared Hoagland working solution is added, and placed in an incubator for cultivation for 1 week. Here, it should be noted that duckweed cultivation adopts a white rectangular wide-mouth plastic culture pot with the same specifications of 2L, and before cultivating duckweed, it needs to be brushed clean, then exposed to the sun for 24 hours and sterilized with ultraviolet for 24 hours in turn.

[0034] In some embodiments, the duckweed species is D. sparsenerivula or D. oligorrhiza.

[0035] In this embodiment, two species of two duckweeds are preferably cultivated, specifically including Lemna aequinoctialis of the genus Lemna and Landoltia punctata of the genus Landoltia. Among them, Lemna aequinoctialis and Landoltia punctata can grow all year round, have a wide range of adaptability to horizontal pH values, can grow normally in the pH range of 5-9, can adapt to the ecological conditions of biogas slurry, do not require additional fertilization or irrigation, and do not compete with crop production for land.

[0036] S200, mixing the biogas slurry with Hoagland's working solution to prepare duckweed culture solution; wherein the concentration of the biogas slurry is 1.5%-5%, and the concentration of the Hoagland's working solution is 95%-98.5%; In this step, in order to improve the low yield of traditional duckweed culture, especially the high cost of using Hoagland's culture solution alone and the insufficient improvement of protein content, by adding biogas slurry to the prefabricated Hoagland's working solution, not only the amount of Hoagland's working solution is reduced, but also the protein content of duckweed can be significantly increased.

[0037] In addition, since biogas slurry contains a large amount of nitrogen and phosphorus, and the metal ions manganese, iron, and zinc content exceed the standard, direct discharge or partial treatment after discharge will still cause eutrophication of water bodies, large-scale algae breeding, and a sharp drop in water oxygen content, which will have a serious impact on the ecology of rivers and lakes. In this step, by using biogas slurry to cultivate duckweed, on the one hand, the protein content of duckweed can be increased, and on the other hand, duckweed will have a high tolerance to high-concentration nutrients in biogas slurry, and can quickly absorb nitrogen and phosphorus elements, organic pollutants, heavy metals and other harmful substances and convert them into high-quality edible tissues, and quickly reproduce and accumulate protein and starch.

[0038] Specifically, the biogas slurry in this step is preferably pig manure wastewater fermented in a fermentation tank of a pig farm wastewater treatment plant for more than 3 months, such as pig manure wastewater from Dongyuan Dongrui Branch of Dongrui Food Group Co., Ltd. The obtained biogas slurry is placed in a 19L transparent covered bucket, which is sealed with a sealing film after the lid is closed, and stored in a dark environment with an ambient temperature maintained below 25°C for no more than 1 month.

[0039] Furthermore, after obtaining the above-mentioned biogas slurry, the biogas slurry and the Hoagland working solution are mixed in proportion to prepare a duckweed culture solution. Specifically, the concentration of the biogas slurry is preferably 1.5%-5%, and the concentration of the Hoagland working solution is preferably 95%-98.5%. Among them, biogas slurry and Hoagland working solution are added to a 2L culture basin respectively, and a glass rod is used to fully stir evenly so that the total culture solution volume in the culture basin is 1L. It should be noted that mixing the biogas slurry and the Hoagland working solution in the above-mentioned proportion can significantly increase the crude protein content of duckweed compared with culturing duckweed with only the Hoagland basic culture solution. The nitrogen and phosphorus in the biogas slurry synergistically act with the potassium, calcium, magnesium, sulfur, iron, manganese, zinc, copper, molybdenum and boron in the Hoagland working solution to jointly promote the growth of duckweed and protein synthesis.

[0040] In some of the embodiments, the total nitrogen content in the biogas slurry is 980 mg / L, and the total phosphorus content is 90 mg / L.

[0041] The pig farm biogas slurry used in this embodiment contains a large amount of nitrogen, phosphorus, potassium and other elements, as well as various trace elements such as copper, iron, sodium, magnesium, zinc, etc., as well as a large amount of microorganisms and organic matter. The total nitrogen content of the biogas slurry is 980 mg / L, and the total phosphorus content is 90 mg / L. It should be noted that in the cultivation process of duckweed, nitrogen is a key factor in protein synthesis. The biogas slurry in this embodiment is rich in ammonia nitrogen NH4-N and nitric nitrogen NO3-N, which can be directly absorbed and utilized by duckweed to promote the synthesis of amino acids and proteins; phosphorus is a component of ATP, nucleic acids and phospholipids, and participates in energy metabolism and protein synthesis. The phosphate PO43- in the biogas slurry can promote the growth and metabolism of duckweed and enhance the efficiency of protein synthesis.

[0042] In some of the embodiments, when the biogas slurry is mixed with the Hoagland working solution, the concentration of the Hoagland working solution is 95%-98%, and the concentration of the biogas slurry is 2%-5%.

[0043] It should be noted that when the concentration of biogas slurry is 2%-5% and the concentration of Hoagland's working solution is 95-98%, the effect of improving duckweed protein is the best. Compared with duckweed cultured with Hoagland's culture solution alone, the crude protein content of duckweed can be significantly improved. For D. paucivenica, the effect is better when the biogas slurry concentration is 2%-4%; for D. pauciroot, the effect is better when the biogas slurry concentration is 3%-5%.

[0044] S300, after the purified duckweed is added to the duckweed culture solution, it is placed in an incubator with preset culture conditions for culture, to obtain cultured duckweed.

[0045] In this step, the purified duckweed is put into the duckweed culture solution so that the biogas slurry and the Hoagland working solution can act on the duckweed together. It should be noted that the incubator needs to be sprayed with 75% medical alcohol for disinfection before formal use, and then disinfected with ultraviolet light for 24 hours.

[0046] In some of the embodiments, the purified duckweed is put into a duckweed culture solution and then placed in an incubator with preset culture conditions for culture to obtain the cultured duckweed, which includes: obtaining the purified duckweed, removing impurities and drying the water on the leaves and roots in sequence to obtain the duckweed to be cultured; putting the duckweed to be cultured into a culture basin containing a culture solution; wherein the leaf surface of the duckweed to be cultured faces upward; placing the culture basin in an incubator and culturing for 5 days according to the preset culture conditions to obtain the cultured duckweed.

[0047] Specifically, during the duckweed cultivation process, the duckweed purified for one week was fished out of the water with a scoop net less than 1 mm, and the debris on the leaves and roots, especially algae, were washed with tap water to prevent the algae from competing with the duckweed for nutrients. Subsequently, the water on the leaves and roots was dried with absorbent paper. Further, 2 g of fresh duckweed was weighed and put into nutrient solutions with different concentrations of biogas slurry so that the leaf surfaces of the duckweed were facing upward and evenly distributed. Among them, all the duckweed was cultured in an incubator for 5 days to obtain the cultured duckweed.

[0048] In some of the embodiments, when the duckweed to be cultured is placed in a culture basin containing culture solution, the surface area of ​​the duckweed covered by the culture solution is greater than 60%.

[0049] It should be noted that in order to enable duckweed to grow rapidly, in this embodiment, duckweed is preferably covered at 60% of the surface of the duckweed culture solution. Since duckweed will grow rapidly only after reaching a certain initial amount, the large initial coverage area of ​​duckweed can block light from irradiating the water, inhibiting the growth of algae in the water, and preventing algae from competing with duckweed for sunlight and growth space after growth. In this embodiment, by covering duckweed at 60% or more of the culture solution surface, light irradiating the bottom of the water can be effectively reduced, thereby inhibiting the growth of algae.

[0050] In some of the embodiments, the preset culture conditions include: a light duration of 16 hours, a dark duration of 8 hours, a light intensity of 6000LUX, a temperature of 25°C, and a humidity of 75%.

[0051] In this embodiment, the culture conditions of the incubator need to be set to 16 hours of light duration (6:00-22:00), 8 hours of darkness, 6000 LUX of light intensity, 25°C of set temperature, and 75% of set humidity. The purification of duckweed and the formal culture of duckweed are both achieved under the above conditions, without the need to set different purification or culture conditions.

[0052] It should be noted that for the determination of protein content after duckweed cultivation, this application uses a semi-automatic Kjeldahl nitrogen analyzer to determine the crude protein content of duckweed. Among them, before the determination, the duckweed needs to be collected and dried. Specifically, after 5 days of duckweed cultivation, use a scoop net with a pore size of less than 1 mm to fish the duckweed out of the water, and use tap water to rinse off debris on the leaves and roots, especially algae. Subsequently, use absorbent paper to absorb the moisture on the leaves and roots, wrap them with A4 paper, ensure that the duckweed has enough heating area, and mark them. Furthermore, all the collected duckweed is dried with a blower dryer at a drying temperature of 105°C and a drying time of 36 hours.

[0053] The determination of duckweed protein will be described in detail below: 1. Preparation of consumables: absorbent paper; marker pen; mortar; balance; weighing paper; test tube; sealing film, 5 mL pipette and tip, other pipettes and tips, 100 ml measuring cylinder, 150 ml conical flask, plastic dropper.

[0054] 2. Reagents: concentrated sulfuric acid, H2O2, distilled water, NaOH solution (10 mol / L), H3BO3 (boric acid, 20 g / L), methyl red-bromocresol green indicator, 0.02 mol / L sulfuric acid.

[0055] 3. Prepare the sample: Sample crushing: After the duckweed sample is completely dried, take it out and grind it into powder using a mortar. Weigh 0.5 g of powder sample (accurate to 0.0002 g) and place it in a dry digestion tube. 4. Digestion: Mark the digestion tubes and add 5 mL of concentrated sulfuric acid in order; Mixing: Mix the sample with concentrated sulfuric acid; Sealing: Seal the tube mouth with plastic wrap to prevent the concentrated sulfuric acid from volatilizing; Overnight: Leave for more than 6 hours, add concentrated sulfuric acid in the afternoon of the previous day, and proceed to the next step in the morning of the next day; Add H2O2: Use a 5 mL pipette to add 5 mL of H2O2 to each test tube. Shake to allow for complete reaction and to turn the solution colorless or clear. Digestion: Place the digestion tube on the digestion furnace (turn on the tap and power supply), the digestion temperature is 370℃, and the digestion time is 10 minutes; after the first digestion, take it down and put it in the fume hood to cool for about 10 minutes, change another plate of samples, and digest the two plates crosswise; add H2O2 before digestion; there is no limit to the number of digestions, and the liquid can be clarified after digestion.

[0056] Ventilation: After disinfection, ventilate in a fume hood for 4 hours; Make up to volume: After digestion, add distilled water to make up to 100 mL.

[0057] Note: H2O2 should be added along the tube wall and a blank control should be set up.

[0058] 5. Nitrogen determination: Sampling: Take 10 mL of the sample after volume adjustment into a new test tube and wait for loading on the machine; Titration bottle: Take 10 ml H3BO3 (boric acid) in a 150 mL conical flask, and then add 2 drops of methyl red-bromocresol green indicator; Clean the machine before and after use: program water 100 mL, boric acid 0, alkali 0, water 10; clean once with ultrapure water; Procedure for use: water 10 mL, boric acid 0, base 10 mL; Start up: Turn on the two switches on the right and left sides of the machine --- drain waste water, exhaust --- place sample --- place titration bottle ------ (auto-start-confirm) Titration: After the machine is finished running, take out the conical flask and titrate with 0.02 mol / L sulfuric acid until it turns transparent red (record the volume of sulfuric acid added) Calculation of results: Calculate the results according to the following formula. The crude protein in the sample is expressed as mass fraction ω, and the value is expressed as mass percentage (%).

[0059] Where: V2-the volume of hydrochloric acid standard titration solution consumed by the titration sample, in milliliters (mL); V1-the volume of the hydrochloric acid standard titration solution consumed in the titration blank, in milliliters (mL); c-concentration of hydrochloric acid standard titration solution, in moles per liter (mol / L); m-sample mass, in grams (g); V-total volume of sample digestion solution, in milliliters (mL); V'- volume of digestion liquid for distillation, in milliliters (mL); 14- molar mass of nitrogen in grams per mole (g / mol); 6.25-average coefficient for conversion of nitrogen into crude protein; Take two parallel samples for each test sample and measure them. The arithmetic mean is taken as the test result. The calculated result is expressed to two decimal places.

[0060] Five groups of examples will be provided below for D. sparsely veined and D. oligorrhizae to further illustrate the method of the present application.

[0061] 1. Duckweed; Example 1 (1) Obtain Lemna sparsenature, wash away debris on the leaves and roots, place it in a culture pot, add Hoagland's working solution, and culture it in an incubator for 1 week to obtain purified Lemna sparsenature; (2) mixing 98.5% of Hoagland's working solution and 1.5% of biogas slurry to obtain a first duckweed culture solution; (3) After washing the purified D. sparse-veined, the debris on the leaves and roots is removed, and the water is absorbed. The D. sparse-veined is placed in the first duckweed culture solution, with the leaf surfaces of the D. sparse-veined facing upward and evenly distributed. The D. sparse-veined covers 60% of the surface of the first duckweed culture solution. The D. sparse-veined is cultured in an incubator for 5 days to obtain the first D. sparse-veined.

[0062] Example 2 Different from Example 1, the proportions of Hoagland's working solution and biogas slurry in Example 2 are 98% and 2% respectively, thereby cultivating the second sparse-veined duckweed.

[0063] Example 3 Different from Example 1, the proportions of Hoagland's working solution and biogas slurry in Example 3 are 97% and 3% respectively, thereby cultivating the third sparse-veined duckweed.

[0064] Example 4 Different from Example 1, the proportions of Hoagland's working solution and biogas slurry in Example 4 are 96% and 4% respectively, thereby cultivating the fourth sparse-veined duckweed.

[0065] Example 5 Different from Example 1, the proportions of Hoagland's working solution and biogas slurry in Example 5 are 95% and 5% respectively, thereby cultivating Duckweed of the fifth rare vein.

[0066] Comparative Example 1 The difference from Example 1 is that only Hoagland working solution is added, and no biogas slurry is added.

[0067] The results of protein content detection of Lemna paucifolia cultured in Examples 1-5 and Comparative Example 1 are as follows: Figure 2 As shown, specifically, when only Hoagland's working solution was used to culture duckweed in Comparative Example 1, the crude protein content in duckweed was 24.18%, while the crude protein content of duckweed in Examples 1-5 after adding biogas slurry increased by 0.08%, 7.65%, 4.79%, 4.61% and 3.08%, respectively. Among them, when the biogas slurry concentration was 3% and 4%, the crude protein content of duckweed was significantly higher than that of duckweed in Comparative Example 1, and when the biogas slurry concentration was 2%, the crude protein content of duckweed was extremely significantly higher than that of duckweed in Comparative Example 1.

[0068] 2. Spirogyra Example 6 (1) Obtain Spirodrum oligorhiza, wash away debris on the leaves and roots, place it in a culture pot, add Hoagland's working solution, and culture it in an incubator for 1 week to obtain purified Spirodrum oligorhiza; (2) mixing 98.5% of Hoagland's working solution and 1.5% of biogas slurry to obtain a first duckweed culture solution; (3) After washing the purified Spirodrum oligorrhizae to remove debris on the leaves and roots, absorb the water and place the Spirodrum oligorrhizae into the first duckweed culture solution, with the leaves of Spirodrum oligorrhizae facing upward and evenly distributed, and the Spirodrum oligorrhizae covering 60% of the surface of the first duckweed culture solution. The Spirodrum oligorrhizae are cultured in an incubator for 5 days to obtain the first Spirodrum oligorrhizae.

[0069] Example 7 Different from Example 6, the proportions of Hoagland's working solution and biogas slurry in Example 7 were 98% and 2% respectively, thereby cultivating the second few-rooted Spirodendrum truncatum.

[0070] Example 8 Different from Example 6, the proportions of Hoagland's working solution and biogas slurry in Example 8 were 97% and 3% respectively, thereby cultivating the third few-rooted Spirodendrum truncatum.

[0071] Example 9 Different from Example 6, the proportions of Hoagland's working solution and biogas slurry in Example 9 were 96% and 4% respectively, thereby cultivating the fourth few-rooted Spirodendrum truncatum.

[0072] Example 10 Different from Example 6, the proportions of Hoagland's working solution and biogas slurry in Example 10 were 95% and 5% respectively, thereby cultivating the fifth Spirodendrum oligophylla.

[0073] Comparative Example 2 The difference from Example 6 is that only Hoagland working solution is added, and no biogas slurry is added.

[0074] The protein content test results of Spirodela oligorrhiza cultured in Examples 6-10 and Comparative Example 2 are as follows: Figure 3 As shown, specifically, when only Hoagland's working solution was used to culture duckweed in Comparative Example 2, the crude protein content in Spirodendrum oligorrhiza was 18.13%, while the crude protein content of duckweed in Examples 6-10 after adding biogas slurry increased by 0.62%, 1.57%, 5.13%, 6.05% and 5.37%, respectively. Among them, when the biogas slurry concentration was 3% and 5%, the crude protein content of duckweed was significantly higher than that of duckweed in Comparative Example 1, and when the biogas slurry concentration was 4%, the crude protein content of duckweed was extremely significantly higher than that of duckweed in Comparative Example 1.

[0075] It can be seen that the method of the present application can significantly increase the protein content of the cultured duckweed by adding biogas slurry to the Hoagland working solution, which can not only efficiently utilize the biogas slurry in the pig farm and save water resources, but also provide an ideal way to recycle aquaculture wastewater and reduce sewage treatment costs. At the same time, the nitrogen and phosphorus in the biogas slurry can be used to promote the synthesis of duckweed protein, enhance the efficiency of protein synthesis, reduce the use of nutrient solution or even completely replace the nutrient solution to obtain stable and reliable duckweed feed by culturing duckweed in large quantities in production practice, providing a good foundation for the research on duckweed as feed.

[0076] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but the limitation of the scope of the invention patent cannot be understood as a result. It should be pointed out that, for a person of ordinary skill in the art, several modifications or improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for increasing duckweed protein, characterized in that: The steps include: Purifying duckweed in Hoagland's working solution to obtain purified duckweed; Mixing the biogas slurry with the Hoagland working solution to prepare duckweed culture solution; wherein the concentration of the biogas slurry is 1.5%-5%, and the concentration of the Hoagland working solution is 95%-98.5%; After the purified duckweed is added to the duckweed culture solution, it is placed in a culture box with preset culture conditions for culture to obtain cultured duckweed.

2. The method for increasing duckweed protein according to claim 1, characterized in that: The Hoagland working solution comprises the following components: Magnesium sulfate heptahydrate 49.2 μg / L, calcium nitrate tetrahydrate 108.6 μg / L, potassium dihydrogen phosphate 27.2 μg / L, potassium nitrate 50.2 μg / L, boric acid 0.286 μg / L, manganese chloride tetrahydrate 0.186 μg / L, zinc sulfate heptahydrate 0.022 μg / L, sodium molybdate dihydrate 0.009 μg / L, copper sulfate pentahydrate 0.009 μg / L, iron (II) sulfate heptahydrate 1.98 μg / L and disodium ethylenediaminetetraacetic acid 6 μg / L.

3. The method for increasing duckweed protein according to claim 2, characterized in that: The Hoagland working solution is obtained by adding pure water to the Hoagland reserve solution to make up the volume; wherein the Hoagland reserve solution comprises the following components: Magnesium sulfate heptahydrate 49.2 g / L, calcium nitrate tetrahydrate 108.6 g / L, potassium dihydrogen phosphate 27.2 g / L, potassium nitrate 50.2 g / L, boric acid 2.86 g / L, manganese chloride tetrahydrate 1.86 g / L, zinc sulfate heptahydrate 0.22 g / L, sodium molybdate dihydrate 0.09 g / L, copper sulfate pentahydrate 0.09 g / L, iron (II) sulfate heptahydrate 1.98 g / L and disodium ethylenediaminetetraacetic acid 6 g / L.

4. The method for increasing duckweed protein according to claim 1, characterized in that: The method of purifying duckweed in Hoagland's working solution to obtain purified duckweed includes: Obtaining duckweed and removing debris from the duckweed to obtain pretreated duckweed; The pretreated duckweed was placed in a culture pot, and the Hoagland working solution was added for purification for 1 week to obtain purified duckweed.

5. The method for increasing duckweed protein according to claim 4, characterized in that: The species of duckweed are D. sparsely veined or D. oligorrhiza.

6. The method for increasing duckweed protein according to claim 1, characterized in that: When the biogas slurry is mixed with the Hoagland working solution, the concentration of the Hoagland working solution is 95%-98%, and the concentration of the biogas slurry is 2%-5%.

7. The method for increasing duckweed protein according to any one of claims 1 to 6, characterized in that: The biogas slurry has a total nitrogen content of 980 mg / L and a total phosphorus content of 90 mg / L.

8. The method for increasing duckweed protein according to claim 1, characterized in that: The method of adding the purified duckweed to the duckweed culture solution and placing the duckweed in a culture box with preset culture conditions for culture to obtain the cultured duckweed comprises: Obtaining the purified duckweed, removing impurities and sucking out moisture from leaves and roots in sequence to obtain duckweed to be cultivated; Putting the duckweed to be cultured into a culture basin containing the culture solution; wherein the leaf surface of the duckweed to be cultured faces upward; The culture pot is placed in the culture box and cultured for 5 days according to the preset culture conditions to obtain cultured duckweed.

9. The method for increasing duckweed protein according to claim 8, characterized in that: When the duckweed to be cultured is put into a culture basin containing the culture solution, the surface area of ​​the duckweed covered by the culture solution is greater than 60%.

10. The method for culturing duckweed protein according to claim 8, characterized in that: The preset culture conditions include: The light duration is 16h, the dark duration is 8h, the light intensity is 6000LUX, the temperature is 25℃ and the humidity is 75%.

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