Method for decolorizing duckweed thalli

Abstract

The invention discloses a novel decoloring method for strong-binding-state duckweed thalli, and belongs to the technical field of chlorophyll removal, and the method comprises the following steps: mixing the duckweed thalli with a decoloring solution according to a ratio of 3-10g / mL, adding calcium carbonate particles, magnetically stirring at 550-750r / min, reacting at 30-70 DEG C for 9-15h, and carrying out one-time liquid change treatment after 3-5h, so that chlorophyll can be removed; a preparation method of the decolorizing solution comprises the step of mixing DMF (Dimethyl Formamide) and ethanol with the concentration of 65-85%. According to the method, high-content chlorophyll in the duckweed thalli can be simply, safely and effectively removed, the decolorized waste liquid can be used for preparing sodium copper chlorophyllin and other products, in addition, ethyl alcohol in the decolorized waste liquid can be recycled after being properly treated, and the experiment cost and the environmental influence are reduced; the decolored duckweed thallus can be used for extraction and preparation of starch, protein, medicinal compounds and the like, and related products can be used in the aspects of biological energy sources, animal feed, sewage treatment, food additives and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of chlorophyll removal, and specifically relates to a method for decolorizing duckweed thalli. Background Art

[0002] Chlorophyll is a key pigment for duckweed to carry out photosynthesis. It can absorb solar energy and convert it into chemical energy for synthesizing organic substances such as glucose, providing energy and material basis for the growth and development of plants. Chlorophyll is a magnesium porphyrin compound with a relatively high color value (greater than 500), and trace amounts can present an obvious green color. The presence of chlorophyll will seriously affect the color and flavor of duckweed end products; and chlorophyll may produce harmful substances under certain conditions, increasing the food safety risk. Duckweed is a widely distributed aquatic floating plant, mostly growing in still waters such as swamps and lakes. Duckweed has a wide growth range, fast growth rate, and short growth cycle, and can reproduce one generation within 2 - 3 days under suitable conditions. The duckweed thalli have a short growth cycle, large yield, and are easily obtained. The thalli of duckweed have a relatively high degree of binding tightness, which is related to its growth characteristics. The thalli of duckweed are composed of a layer of epidermal cells and internal mesophyll cells, and a large number of cell gaps are formed between the cells. This structure enables duckweed to float on the water surface and carry out effective photosynthesis. However, this structure also makes it difficult for external substances, including decolorizing agents, to penetrate into the thalli, thus increasing the difficulty of decolorization. In addition, the surface of duckweed has a layer of wax, which can protect duckweed from the harm of the external environment and also hinders the penetration of decolorizing agents.

[0003] The following is an example of a description of the publicly disclosed representative technical inventions:

[0004] (1) A method for removing microalgae chlorophyll (Patent Application No.: CN201410828946.7). Compared with algae, duckweed is more difficult to decolorize due to its structural differences (the thalli of duckweed are tightly bound and have more cell gaps, which makes it difficult for external substances to penetrate into the thalli), surface characteristics (the surface of duckweed has a layer of wax, which can protect duckweed from the harm of the external environment and also hinders the penetration of decolorizing agents), growth characteristics (the growth rate of duckweed is very fast, and it has low requirements for water quality and can be cultivated in slightly soft to slightly hard fresh water. This fast growth characteristic may affect the effect of decolorizing agents), and environmental adaptability (duckweed has strong environmental adaptability and can grow under various water quality conditions. This adaptability may make it have a certain resistance to decolorizing agents).

[0005] (2) A method for decolorizing DAB - stained plant leaves (Patent Application No.: CN201610396280.1). However, this technology has complex operations, and the strong decolorizing solution used may involve harmful chemical reagents, posing certain environmental and health risks. In addition, the decolorization step after DAB staining is for DAB to H2 O 2 The test of the accuracy of staining indication. At present, the method of decolorization after DAB staining has low resolution, and the decolorization effect is not very significant. Often, the miscellaneous colors cannot be completely removed;

[0006] (3) The composition and method for enzymatic decolorization of chlorophyll (Patent Application No.: CN201310031421.6). The cost of this technology is relatively high, and it is still in the laboratory exploration stage at present. At the same time, the raw material treated is the extract of chlorophyll, and it cannot directly decolorize the duckweed thalli;

[0007] (4) A decolorization column and a method for normal-temperature decolorization of DHA oil by using the same (Patent Application No.: CN201010227935.5). However, the cost of scaling up this technology is relatively high, and it is difficult to be applied on a large scale. At the same time, this technology cannot process duckweed thalli with a high chlorophyll content.

[0008] In summary, most of the currently disclosed decolorization technologies have certain limitations and lack a decolorization process specifically for duckweed thalli with high binding tightness. Summary of the Invention

[0009] This patent provides a simple, safe, effective and low-cost decolorization process for duckweed thalli, effectively solving the problem of chlorophyll pollution in duckweed products. The decolorization waste liquid can be used to prepare products such as sodium copper chlorophyllin. The first object of the present invention is to provide a method for decolorizing duckweed thalli, and the method includes the following steps:

[0010] Step 1: Mix the duckweed thalli with the decolorizing solution at a material concentration of 3-10%, add calcium carbonate particles, stir magnetically at 550-750 r / min, continuously react at 30-70 °C for 3-5 h, pour out the waste liquid, and rinse to obtain the duckweed thalli after the first decolorization;

[0011] Step 2: Mix the duckweed thalli after the first decolorization with the decolorizing solution, and continue to react at 30-70 °C for 4-12 h, then rinse;

[0012] Step 3: Freeze-dry to obtain the de-greened duckweed thalli powder;

[0013] The decolorizing solution is composed of dimethylformamide (DMF) and ethanol with a concentration of 65-85% in a volume ratio of 1:2-4.

[0014] DMF is a high-boiling point, colorless, distillable liquid with good solubility, capable of dissolving a variety of organic compounds. Ethanol is also a commonly used organic solvent. When the two are used in combination, their solvent characteristics can be complementary, improving the solubility of chlorophyll. The addition of calcium carbonate also helps to break the cell structure, making the grinding more thorough, thus releasing more pigments. Chlorophyll is easily hydrolyzed by chlorophyllase in living cells. The addition of calcium carbonate can neutralize the acidic environment in the cells, thereby inhibiting the activity of chlorophyllase and reducing pigment degradation. Calcium carbonate particles physically adsorb chlorophyll molecules on their surface, while the decolorizing solution evenly distributes chlorophyll molecules in the solution through dissolution. This synergistic effect increases the residence time of chlorophyll molecules in the solution and the contact opportunities with calcium carbonate particles, thus improving the decolorization efficiency.

[0015] In one embodiment, the induced duckweed is obtained by culturing the duckweed expanded in Hoagland nutrient solution under white light and deionized water for 48 h.

[0016] In one embodiment, the addition amount of the calcium carbonate particles in step 1 is 5-10 wt% of the wet basis of duckweed thalli.

[0017] In one embodiment, the rinsing is carried out with deionized water until the rinsing liquid is completely colorless and transparent.

[0018] In one embodiment, the duckweed includes Spirodela polyrhiza, Spirodela oligorrhiza or Lemna minor.

[0019] In one embodiment, the lyophilization is carried out after pre-freezing in a -80 °C refrigerator for 12 h and then performing lyophilization for 48 h.

[0020] In one embodiment, DMF and 80% ethanol are mixed in a volume ratio of 1:2.

[0021] In one embodiment, duckweed thalli and the decolorizing solution are mixed at a ratio of 1:15 g / mL.

[0022] In one embodiment, the stirring rate is 650 r / min.

[0023] In one embodiment, the reaction temperature in step 1 is 40 °C and the reaction time is 4 h. In one embodiment, the reaction temperature in step 1 is 50 °C and the reaction time is 4 h.

[0024] In one embodiment, the reaction temperature in step 1 is 60 °C and the reaction time is 4 h.

[0025] In one embodiment, the reaction temperature in step 2 is 40 °C and the reaction time is 8 h.

[0026] In one embodiment, the reaction temperature in step 2 is 50 °C and the reaction time is 7 h.

[0027] In one embodiment, the reaction temperature in step 2 is 60 °C and the reaction time is 8 h.

[0028] The second object of the present invention is to provide further utilization of the decolorized waste liquid obtained after decolorizing the duckweed thalli in the preparation of sodium copper chlorophyll products.

[0029] The third object of the present invention is to provide the application of the de-greened duckweed thalli powder obtained after decolorizing the above-mentioned duckweed thalli in the extraction and preparation of starch, protein, medicinal compounds, etc.

[0030] Beneficial effects

[0031] The present invention uses duckweed as the main raw material. The duckweed decolorization technology is of great significance for improving its application value in multiple fields, including environmental protection, resource utilization, bioenergy development, medicinal value, and food industry, etc.

[0032] The present invention can use the decolorized liquid to prepare sodium copper chlorophyll, achieving the recycling of waste liquid. The present invention can effectively remove chlorophyll from duckweed thalli. The decolorized duckweed thalli can be used for the extraction and preparation of starch, protein, medicinal compounds, etc. The related products can be used in aspects such as bioenergy, animal feed, sewage treatment, food additives, etc. Decolorization will greatly improve the flavor and quality of the products. Description of the drawings

[0033] Figure 1 It is a comparison chart of three examples and four comparative examples.

[0034] Figure 2 It is the influence of different factors on the decolorization degree. A is the decolorization time; B is the liquid replacement time; C is the ratio of material to liquid; D is the temperature; E is the ethanol concentration; F is the magnetic stirring rate. Specific embodiments

[0035] Example 1

[0036] Select the fresh Wolffia arrhiza thalli induced for 48 h under oligotrophic conditions as the raw material. The leaves before decolorization are green or yellowish-green. Through the optimization of process parameters, the optimal decolorization conditions are obtained: mix the Wolffia arrhiza thalli with the decolorizing solution (DMF and 80% ethanol mixed at a ratio of 1:2) according to the solid-liquid ratio (mass-volume ratio) of 1:15 g / mL, add 2% calcium carbonate particles, stir at a rate of 650 r / min, react at 40 °C for 4 h, pour out the waste liquid after the above reaction is completed and rinse it with deionized water until clean, add the same volume of decolorizing solution again and continue to react at 40 °C for 8 h, pour out the waste liquid and rinse several times until there is no residue of the decolorizing solution, pre-freeze in an -80 °C refrigerator for 12 h and then perform freeze-drying for 48 h. After grinding into powder, measure the color difference, as Figure 1 shown.

[0037] Example 2

[0038] Select the fresh Wolffia arrhiza thalli induced for 48 h under oligotrophic conditions as the raw material. The leaves before decolorization are green or yellowish-green. Mix the Wolffia arrhiza thalli with the decolorizing solution (DMF and 85% ethanol mixed at a ratio of 1:2) according to the solid-liquid ratio (mass-volume ratio) of 1:15 g / mL, add 2% calcium carbonate particles, stir at a rate of 600 r / min, react at 50 °C for 4 h, pour out the waste liquid after the above reaction is completed and rinse it with deionized water until clean, add the same volume of decolorizing solution again and continue to react at 50 °C for 7 h, pour out the waste liquid and rinse several times until there is no residue of the decolorizing solution, pre-freeze in an -80 °C refrigerator for 12 h and then perform freeze-drying for 48 h. After grinding into powder, measure the color difference, as Figure 1 shown.

[0039] Example 3

[0040] Select the fresh Wolffia arrhiza thalli induced for 48 h under oligotrophic conditions as the raw material. The leaves before decolorization are green or yellowish-green. Mix the Wolffia arrhiza thalli with the decolorizing solution (DMF and 80% ethanol mixed at a ratio of 1:2) according to the solid-liquid ratio (mass-volume ratio) of 1:15 g / mL, add 2% calcium carbonate particles, stir at a rate of 700 r / min, react at 60 °C for 4 h, pour out the waste liquid after the above reaction is completed and rinse it with deionized water until clean, add the same volume of decolorizing solution again and continue to react at 60 °C for 8 h, pour out the waste liquid and rinse several times until there is no residue of the decolorizing solution, pre-freeze in an -80 °C refrigerator for 12 h and then perform freeze-drying for 48 h. After grinding into powder, measure the color difference, as Figure 1 shown.

[0041] Comparative Example 1

[0042] The powder of non-decolorized Wolffia arrhiza thalli is prepared in this comparative example. The specific operation steps are as follows: Select the fresh Wolffia arrhiza thalli induced for 48 h under oligotrophic conditions as the raw material, spin-dry the water, pre-freeze in an -80 °C refrigerator for 12 h and then perform freeze-drying for 48 h. After grinding into powder, measure the color difference, asFigure 1 as shown

[0043] Comparative Example 2

[0044] Fresh duckweed thalli induced for 48 h under oligotrophic conditions were selected as raw materials. The leaves before decolorization were green or yellowish-green. The duckweed thalli were mixed with a decolorizing solution (80% ethanol) at a solid-liquid ratio (mass-to-volume ratio) of 1:15 g / mL, with a stirring rate of 650 r / min, and reacted at 40 °C for 4 h. The waste liquid after the above reaction was poured out and rinsed clean with deionized water. The same volume of the decolorizing solution was added again and the reaction continued at 40 °C for 8 h. The waste liquid was poured out and rinsed several times until no decolorizing solution remained. After pre-freezing in a -80 °C refrigerator for 12 h, freeze-drying was carried out for 48 h. After grinding into powder, the color difference was measured, as Figure 1 as shown

[0045] Comparative Example 3

[0046] Fresh duckweed thalli induced for 48 h under oligotrophic conditions were selected as raw materials. The leaves before decolorization were green or yellowish-green. The duckweed thalli were mixed with a decolorizing solution (a mixture of DMF and 80% ethanol in a ratio of 1:2) at a solid-liquid ratio (mass-to-volume ratio) of 1:15 g / mL, with a stirring rate of 650 r / min, and reacted at 40 °C for 4 h. The waste liquid after the above reaction was poured out and rinsed clean with deionized water. The same volume of the decolorizing solution was added again and the reaction continued at 40 °C for 8 h. The waste liquid was poured out and rinsed several times until no decolorizing solution remained. After pre-freezing in a -80 °C refrigerator for 12 h, freeze-drying was carried out for 48 h. After grinding into powder, the color difference was measured, as Figure 1 as shown

[0047] Comparative Example 4

[0048] Fresh duckweed thalli induced for 48 h under oligotrophic conditions were selected as raw materials. The leaves before decolorization were green or yellowish-green. The duckweed thalli were mixed with a decolorizing solution (80% ethanol) at a solid-liquid ratio (mass-to-volume ratio) of 1:15 g / mL, and 2% calcium carbonate particles were added. The stirring rate was 650 r / min, and the reaction was carried out at 40 °C for 4 h. The waste liquid after the above reaction was poured out and rinsed clean with deionized water. The same volume of the decolorizing solution was added again and the reaction continued at 40 °C for 8 h. The waste liquid was poured out and rinsed several times until no decolorizing solution remained. After pre-freezing in a -80 °C refrigerator for 12 h, freeze-drying was carried out for 48 h. After grinding into powder, the color difference was measured, as Figure 1 as shown

[0049] Table 1 Brightness, yellow-blue chromaticity, red-green chromaticity values of three examples and four comparative examples and their change values compared with the blank dish

[0050]

[0051]

[0052] As Figure 2 shown, according to preliminary experiments, the decolorization time, liquid change time, material-liquid ratio, temperature, ethanol concentration, and stirring rate have the greatest impact on the decolorization degree. According to the experiments, the optimal decolorization conditions are as follows: Mix the duckweed thalli with the decolorizing solution (DMF and 80% ethanol mixed at a ratio of 1:2) at a material concentration (mass-volume ratio) of 1:15 g / mL, stir at a rate of 650 r / min, react at 40 °C for 12 h, and perform a liquid change treatment every 4 h to remove chlorophyll to the greatest extent.

[0053] The decolorization time is set to 12 h, and a liquid change treatment is performed every 4 h. This operation maintains the effectiveness of the decolorizing solution during the long decolorization time and can remove impurities or saturated decolorizing agents generated during the reaction, thus maintaining the high efficiency of the decolorizing solution. Temperature and stirring rate have a significant impact on the rate and efficiency of the decolorization reaction. The optimal temperature of 80 °C can accelerate the contact and reaction between pigment molecules and the decolorizing agent to the greatest extent; stirring can ensure sufficient contact between the duckweed thalli and the decolorizing solution, avoiding local concentration being too high or too low. At the same time, an ethanol concentration of 80% may help dissolve pigment molecules, making them more easily accessible to the decolorizing agent, thereby improving the decolorization effect. The material-liquid ratio (mass-volume ratio) of 1:15 g / mL ensures sufficient contact between the decolorizing solution and the duckweed thalli while avoiding waste of the decolorizing solution. The synergy between temperature and stirring rate can accelerate the decolorization process, while the liquid change operation further maintains the high efficiency of the decolorizing solution. The experimental results show that there is a significant synergy among these factors.

[0054] The strongly polar DMF and the moderately polar ethanol form a dynamic polar gradient environment. The high-concentration ethanol of 80% promotes penetration by disrupting the phospholipid bilayer of the cell membrane (similar solubility principle). DMF, as a phase transfer catalyst, targets and deconstructs the chlorophyll porphyrin ring through strong hydrogen bond interactions, promoting the migration of lipophilic chlorophyll from the cell matrix to the solvent phase. After DMF dissolves the pigment molecules, calcium carbonate can more effectively adsorb these dissolved pigment molecules due to its microporous structure. In addition, the CO3 2- produced by the decomposition of calcium carbonate forms a buffer system to maintain a pH of 8 - 9 (the optimal condition for alkaline demetallation of chlorophyll), providing a stable environment for the dissolution of DMF and preventing chemical changes of pigment molecules under acidic conditions. The combination of the dissolution of DMF and the adsorption of calcium carbonate better disperses pigment molecules, thereby improving the decolorization efficiency.

[0055] Ethanol (boiling point 78 °C) and DMF (boiling point 153 °C) form an azeotropic system after mixing (actual boiling point is about 85 - 90 °C). 40 °C is close to the glass transition temperature of ethanol (~45 °C), which may soften the cell wall structure and enhance solvent penetration. Such an operating temperature (far below the azeotropic point) can reduce solvent volatilization loss. At the same time, stirring (650 r / min) promotes gas-liquid equilibrium and maintains the stability of the solvent ratio. 40 °C promotes partial decomposition of calcium carbonate Released Ca 2+ Displaces Mg in chlorophyll 2+ (The ion displacement rate increases with the increase of temperature), and stirring at 650 r / min accelerates Ca 2+ Mass transfer and shortens the demetallation reaction time. The micropores of calcium carbonate (pore size ~2 - 50 nm) adsorb free chlorophyll (molecular size ~1.5 nm), and the dissolution equilibrium is pushed towards the direction of chlorophyll release by Le Chatelier's principle. When the solid-liquid ratio (mass-volume ratio) is 1:15 g / mL, the sufficient solvent volume delays the saturation of the calcium carbonate adsorption sites.

[0056] The above are only the embodiments of the present invention, and thus do not limit the patent scope of the present invention. All equivalent transformations made using the content of the specification of the present invention, directly or indirectly applied in related technical fields, are similarly included in the patent protection scope of the present invention.

Claims

1. A method for decolorizing duckweed fronds, characterized in that: The method comprises the following steps: Step 1, mixing duckweed fronds with a decolorizing solution at a material concentration of 3-10%, adding calcium carbonate particles, magnetically stirring at 550-750 r / min, reacting at 30-70° C. for 3-5 hours, dumping the waste liquid, and washing to obtain the duckweed fronds after the first decolorization; Step 2, mixing the duckweed fronds after the first decolorization with the decolorization solution, continuing the reaction at 30-70° C. for 4-12 hours, and washing; Step 3, freeze-drying to obtain dechlorotic duckweed frond powder; The decolorizing solution is a mixture of dimethylformamide and 65-85% ethanol in a volume ratio of 1:2-4.

2. The method for decolorizing duckweed fronds according to claim 1, characterized in that: The duckweed is prepared by culturing the duckweed expanded with Hoagland nutrient solution under white light deionized water for 48 hours.

3. The method for decolorizing duckweed fronds according to claim 1, characterized in that: The amount of calcium carbonate particles added in step 1 is 5-10wt% of the wet basis of duckweed fronds.

4. The method for decolorizing duckweed fronds according to claim 1, characterized in that: The washing is to wash the leaf blade with deionized water until the washing liquid is completely colorless and transparent.

5. The method for decolorizing duckweed fronds according to claim 1, characterized in that: The duckweed includes Spirodendrum multirhizum, Spirodendrum oligorhizum or Spirodendrum natans.

6. The method for decolorizing duckweed fronds according to claim 1, characterized in that: The freeze drying is performed by pre-freezing in a -80°C refrigerator for 12 hours and then freeze drying for 48 hours.

7. The decolorized waste liquid obtained by the decolorization method of duckweed fronds according to any one of claims 1 to 6 can be further utilized in the preparation of sodium copper chlorophyllin products.

8. The dechlorotic duckweed frond powder obtained by the decolorization method of duckweed fronds according to any one of claims 1 to 6 is used in the extraction and preparation of starch, protein, medicinal compounds, etc.

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