Method for extracting chlorophyll from medicago sativa hay

Abstract

The invention discloses a method for extracting chlorophyll from medicago sativa hay. The method comprises the following steps: drying the medicago sativa hay until the water content is lower than 20%, and crushing the medicago sativa hay to 80 meshes to obtain hay powder; pressing and rehydrating the grass meal, performing percolation extraction by taking alcohol as a solvent to obtain a brown yellow extracting solution and a green extracting solution, and performing vacuum concentration on the obtained green extracting solution at 60 DEG C to obtain chlorophyll crude paste with the color value of more than 60; performing vacuum concentration on the brown yellow extracting solution at 60 DEG C to obtain water-soluble protein crude paste; the rehydration operation comprises the steps that pressed grass cakes or grass balls are added into a sodium carbonate solution to be soaked for 1 hour, and the pH value of the solution in the rehydration process is alkaline. The color value of the chlorophyll extracted by the method is far higher than that of chlorophyll extracted from silkworm excrement, the chlorophyll has fewer impurities, and the green color of the raw materials used after extraction can be completely faded, so that the method is particularly suitable for extracting the chlorophyll contained in alfalfa, the extraction rate can reach 8.57 mg / g, and the color value is 60 or more.

Description

Technical Field

[0001] The present application relates to the technical field of alfalfa dry powder extraction, and in particular to a method for extracting chlorophyll from alfalfa. Background Art

[0002] Traditional chlorophyll extraction mainly uses acetone as a solvent to extract from silkworm feces. The color value of the extracted chlorophyll crude paste is around 25, and it contains many impurities. To meet the requirements of commercial application, the crude paste needs to be subjected to multiple fine extraction operations. The process is cumbersome and the product loss is serious, resulting in a low final yield, which cannot meet the requirements of industrial large-scale production.

[0003] Alfalfa is a perennial herbaceous plant belonging to the Fabaceae family, Fabaceae subfamily, Trifoliaceae tribe, Medicago genus. It is one of the most widely cultivated legumes in the world, mainly distributed in the Mediterranean region, Africa, Southwest Asia, and Central Asia. Currently, my country's alfalfa cultivation area ranks among the highest in the world, with 13 species and 1 variety widely distributed in the Huanghuaihai region, North China, Northwest China, and Northeast China, mainly concentrated in Shaanxi, Shanxi, and Gansu provinces. Alfalfa is mainly used as livestock feed, processed into primary products such as bales, grass blocks, grass meal, or grass pellets. The active ingredients reported in alfalfa include flavonoids, polysaccharides, saponins, organic acids, coumarins, and proteins.

[0004] Due to the low chlorophyll content in alfalfa and the fact that the chlorophyll content of alfalfa hay is further reduced due to factors such as reduced moisture content and heating and drying, there have been no relevant research reports on using it as a raw material for extracting chlorophyll.

[0005] Although grass plants are low-cost, they contain a large amount of plant fiber and protein in addition to chlorophyll. Existing extraction methods cannot separate chlorophyll from other components during the extraction stage, resulting in the obtained chlorophyll having a low color value and a high impurity content. In addition, the combination of cellulose, protein and chlorophyll is chemical bonded, requiring a large number of purification operations to achieve separation, and the yield of the product after separation is also low.

[0006] In addition to the commonly used solvent extraction method, there are also microwave extraction and ultrasonic extraction methods for chlorophyll extraction. However, each method has different problems:

[0007] 1) Ultrasonic extraction is limited by ultrasonic attenuation. To avoid the formation of ultrasonic blank zones within the container, the extraction equipment must be small. Due to the volume of the extraction container, the extraction process can only be carried out in batches, making continuous large-scale industrial production impossible.

[0008] 2) Microwaves are non-ionizing electromagnetic radiation with limited penetration depth (on the same order of magnitude as their wavelength), but their mass transfer effect in the enhanced extraction process is not obvious. To ensure the penetration depth of microwaves, the capacity of the microwave equipment currently used is small and cannot achieve continuous large-scale industrial production.

[0009] In addition, although microwave extraction and ultrasonic extraction have high extraction rates, the equipment used is relatively expensive and the equipment maintenance cost is high. Summary of the Invention

[0010] The present application provides a method for extracting chlorophyll from alfalfa, which is used to solve the technical problems existing in the prior art of industrial chlorophyll extraction methods, such as low color value of crude paste obtained, high impurity content, cumbersome extraction operation, and reduced product yield; and ultrasonic and microwave batch processing.

[0011] The present application provides a method for extracting chlorophyll from alfalfa, comprising the following steps: drying the alfalfa to a moisture content of less than 20% and then crushing the alfalfa to 80 mesh to obtain grass powder; pressing and rehydrating the grass powder, and then performing percolation extraction with alcohol as a solvent to obtain a brown-yellow extract and a green extract; concentrating the obtained green extract under reduced pressure at 60° C. to obtain a crude chlorophyll paste, wherein the color value of the obtained crude chlorophyll paste is above 60; concentrating the brown-yellow extract under reduced pressure at 60° C. to obtain a crude water-soluble protein paste;

[0012] The rehydration operation is as follows: sodium carbonate solution is added to the compressed grass cake or grass ball, and soaked for 1 hour. The pH value of the solution is alkaline during the rehydration process.

[0013] Rehydration yields approximately 2.15 mg / g, a yield that can increase by over 300%. However, this process generates acid, raising the pH of the extract to around 4.5. The magnesium in the chlorophyll molecules is easily replaced by hydrogen, resulting in pheophytin-free chlorophyll. Using a sodium carbonate solution not only softens the hay powder more quickly but also neutralizes the acid generated during the rehydration process, maintaining an alkaline pH during the rehydration process, protecting the chlorophyll and improving yield and color value. Pressing improves hay compactness and reduces rehydration volume. This method, which sequentially combines drying, milling, pressing, rehydration, and percolation extraction, effectively improves the color value and yield of the crude paste. This method is particularly suitable for large-scale industrial production.

[0014] Preferably, the alfalfa is dried by airing for 2 days or drying at 80°C.

[0015] Preferably, the amount of sodium carbonate added to the sodium carbonate solution is 1-2% of the mass of the grass powder to be processed, and the mass of the aqueous solution used is 3 times the mass of the grass powder to be processed.

[0016] Preferably, the percolation extraction comprises the following steps: discharging the soaking liquid and simultaneously adding 80° alcohol into the extraction device during the discharging process, with the flow rate of the alcohol being the same as the flow rate of the soaking liquid.

[0017] Preferably, the flow rates of the soaking liquid and the alcohol are both 0.8 L / h.

[0018] Preferably, the first 3-4 times the amount of extract during the draining process is a brownish-yellow extract, and the last 6-7 times the amount of extract during the draining process is chlorophyll. Using this method for draining, the grass powder can also absorb excess solvent, reducing the solvent content in the extract.

[0019] Specifically, the method comprises the following steps:

[0020] 1. Select 4000g-6000g of alfalfa, irradiate it under sunlight for 2 days, collect and grind it, select 80 mesh sieve, deduct the residual raw materials in the grinder, and collect alfalfa dry powder with a moisture content of 13.88-15.27%.

[0021] 2. Put the dry powder obtained above into a grass press and press it into grass cakes of 100g each.

[0022] 3. Put the obtained grass cake into the extraction device, weigh 7.7-11.5g of sodium carbonate, dissolve the sodium carbonate with 2300-3450mL of water, add the obtained solution to the extraction column, and infiltrate for one hour.

[0023] 4. Release the soaking liquid at a flow rate of 0.8 L / h and add 80° alcohol into the extraction device simultaneously during the release process. The flow rate of the alcohol is the same as that of the soaking liquid. Collect the dark green extract and concentrate the obtained extract under reduced pressure at 60 degrees Celsius to obtain a crude chlorophyll paste.

[0024] The beneficial effects of this application include:

[0025] 1) The present application provides a method for extracting chlorophyll from alfalfa. The chlorophyll extracted using this method has a color value much higher than that extracted from silkworm excrement, has fewer impurities, and completely removes the green color of the raw material after extraction. This method is particularly suitable for extracting chlorophyll contained in alfalfa, with an extraction rate of up to 8.57 mg / g, a color value of over 60, and a crude chlorophyll content of over 50%. The solvent used is safe and non-toxic, and there is no problem of acetone solvent residue. The method is simple in process, has low equipment investment cost, and is suitable for industrial production.

[0026] 2) The method provided in this application for extracting chlorophyll from alfalfa can obtain a crude chlorophyll paste and an extract containing water-soluble protein at the same time, thereby achieving separation of protein and chlorophyll in alfalfa during the extraction stage without the need for subsequent fine extraction and separation, and the recovered water-soluble protein can also be used in other fields.

[0027] 3) The method for extracting chlorophyll from alfalfa provided in this application adjusts the pH value of the extract by adding sodium carbonate during the rehydration process of the extraction, thereby avoiding chlorophyll denaturation during the extraction process, effectively improving the yield and color value of the obtained chlorophyll, and improving product quality.

[0028] 4) The method for extracting chlorophyll from alfalfa provided in this application is simple to operate, requires low equipment investment, and is particularly suitable for industrial large-scale production. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0030] The technical means that are not described in detail in this application and are not used to solve the technical problems of this application are all set according to the common knowledge in this field, and can be implemented in a variety of common knowledge settings.

[0031] The method for extracting chlorophyll from alfalfa provided in the present application comprises the following steps:

[0032] Example

[0033] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels.

[0034] Example 1

[0035] 1. 4000 g of alfalfa was selected, exposed to sunlight for 2 days, collected and crushed, and a sieve of 80 mesh was selected. After deducting the residue from the crushing machine, a total of 770.8 g of alfalfa dry powder was collected, with a moisture content of 15.27%.

[0036] 2. Put the dry powder obtained above into a grass press and press it into grass cakes of 100g each, totaling 768.5g.

[0037] 3. Put 768.5g of grass cake into the extraction column, weigh 7.7g of sodium carbonate, dissolve the sodium carbonate in 2300mL of water, add the resulting solution into the extraction column, and infiltrate for one hour.

[0038] 4. After one hour, start draining the liquid and control the flow rate to 0.8L / h. At the same time, add 8000mL of 80% alcohol at this flow rate. A total of 3000mL of brown-yellow extract and 5000mL of dark green extract were collected.

[0039] 5. The collected green chlorophyll extract was concentrated under reduced pressure at 60 degrees Celsius to obtain 50.38 g of chlorophyll crude paste. 0.0932 g of the chlorophyll crude paste was diluted with acetone to 18.6219 g. 0.2903 g of the solution was diluted with acetone to 25 ml. The absorbance was measured using UV-2550 and the chlorophyll color value was calculated. The content detection method was based on SN / T 1113-2002 "Determination of phycocyanin and chlorophyll content in Spirulina for import and export". The test results of the samples obtained in this example are shown in the following table: max : Maximum absorption wavelength; A: absorbance value at the maximum absorption wavelength; E: color value;

[0040]

[0041] As can be seen from the above table, the chlorophyll content in the obtained crude chlorophyll paste is 68.43%, and the chlorophyll extraction rate in the raw material can reach 8.57 mg / g. This method can effectively extract the chlorophyll contained in alfalfa.

[0042] After the brown-yellow extract is concentrated according to the above method, the resulting paste is detected as a protein by the Coomassie Brilliant Blue G-250 colorimetric method. It is soluble in water and can be precipitated by acetone at a low concentration, and can be further separated, recovered and utilized.

[0043] Example 2

[0044] 1. 6000 g of alfalfa was selected, exposed to sunlight for 2 days, collected and crushed, and sieved with 80 mesh. After deducting the residue from the crushing machine, a total of 1156.2 g of alfalfa dry powder was collected, with a moisture content of 13.88%.

[0045] 2. Put the dry powder obtained above into a grass press and press it into grass cakes of 100g each, totaling 1152.4g.

[0046] 3. Put 1152.4g of grass cake into the extraction column, weigh 11.5g of sodium carbonate, dissolve the sodium carbonate in 3450ml of water, add the resulting solution into the extraction column, and infiltrate for one hour.

[0047] 4. After one hour, start draining the liquid and control the flow rate to 0.8L / h. At the same time, add 12000mL of 80% alcohol at this flow rate. A total of 3500mL of brown-yellow extract and 7500mL of green extract are collected.

[0048] 5. The collected green chlorophyll extract was concentrated under reduced pressure at 60 degrees Celsius to obtain a total of 75.62 g of chlorophyll crude paste. 0.0431 g of the chlorophyll crude paste was dissolved in acetone to 100 mL and then diluted 5 times. The absorbance was measured using UV-2550 and the chlorophyll color value was calculated. The content detection method was based on SN / T1113-2002 "Determination of Phycocyanin and Chlorophyll Content in Spirulina for Import and Export". The test results of the samples obtained in this example are shown in the following table: max : Maximum absorption wavelength; A: absorbance value at the maximum absorption wavelength; E: color value;

[0049]

[0050] After the brown-yellow extract was concentrated as described above, the resulting paste was detected by Coomassie Brilliant Blue G-250 colorimetric method to determine if it contained some water-soluble protein.

[0051] Example 3: Pilot test of chlorophyll extraction from alfalfa

[0052] 1. Feeding: Select 500kg of dried alfalfa, put it into the grass press, press it into 100g / piece grass cake, collect the grass cakes, and put them directly into the 5m 3 In the extraction tank, a filter bag is added to the liquid discharge port of the extraction tank.

[0053] 2. Rehydration: Add 1500L of pure water, dissolve 5kg of sodium carbonate in the pure water first, put the resulting grass cake into the solution, and soak for 1 hour.

[0054] 3. Percolation extraction: After rehydration, add 80% alcohol to the extraction tank, a total of 5000 kg, and the alcohol is transported through a pipeline with a controlled flow rate of 0.8 L / h. At the same time, the lower end of the extraction tank begins to discharge liquid at a rate consistent with the alcohol flow rate.

[0055] 4. Collect the extract in sections, collect 1800kg of brown-yellow extract, and temporarily place it in a 2t storage tank. When green appears, start collecting it and put it into the transfer tank, which is about 6100kg.

[0056] 5. Concentration: The chlorophyll extract was directly fed from the transfer tank into a double-effect concentration device. When the alcohol content was less than 10%, a crude paste was collected. A total of 6.39 kg of crude paste was obtained. 0.0521 g of the crude chlorophyll paste was dissolved in acetone to 100 mL and then diluted 5 times. The absorbance was measured using a UV-2550 spectrophotometer, and the chlorophyll color value was calculated. The content detection method was based on SN / T 1113-2002 "Determination of Phycocyanin and Chlorophyll Content in Spirulina for Import and Export". The test results of the samples obtained in this example are shown in the following table: max : Maximum absorption wavelength; A: absorbance value at the maximum absorption wavelength; E: color value;

[0057]

[0058] Example 4

[0059] The difference from Example 1 is that the amount of sodium carbonate added to the sodium carbonate solution is 2% of the mass of the grass powder to be processed, and the mass of the aqueous solution used is 3 times the mass of the grass powder to be processed.

[0060] Comparative Example 1;

[0061] Silkworm excrement was used as raw material, crushed to 80 mesh, and acetone was added as an extraction solvent. The resulting solution was extracted by percolation at room temperature and concentrated by the same method as in Example 1 to obtain a crude chlorophyll paste. The color value of the obtained crude chlorophyll paste was about 25, and it contained a lot of impurities.

[0062] Comparative Example 2

[0063] The difference from Comparative Example 1 is that a desolventizing step is added: After concentration until no obvious liquid outflow occurs using the same method as in Example 1, 1 times the amount of water is added to the resulting crude paste and the concentration is continued until no liquid outflow occurs. This operation is repeated once. The purpose of this operation is to replace the acetone mixed in the crude paste with water. The remaining steps are the same as in Comparative Example 1.

[0064] The obtained product was tested for residual solvent acetone by gas chromatography in accordance with GB 5009.262-2016 "National Food Safety Standard - Determination of Residual Solvents in Food". The results showed that after desolventization treatment, the residual acetone decreased by 520 ppm, but was still in a relatively high range.

[0065] Comparative Example 3

[0066] The difference from Example 1 is that sodium carbonate is not added in step 3).

[0067] The parameter results of the products obtained in Examples 1-2 and Comparative Examples 1-2 are shown in the table below:

[0068] serial number raw material <![CDATA[λ max ]]> E Extraction rate mg / g Chlorophyll content of crude paste Acetone residual ppm Comparative Example 1 silkworm feces 663.0 25.06 3.33 24.37％ 1855 Comparative Example 2 silkworm feces 663.5 24.89 2.98 23.96％ 1335 Example 1 Alfalfa 664.5 73.52 8.57 68.43％ - Example 2 Alfalfa 664.5 61.33 7.14 56.65％ -

[0069] As can be seen from the above, the method provided by the present application is suitable for alfalfa with a low chlorophyll content among plants. The color of the alfalfa raw material can be improved from green to colorless. Using dry material extraction, the extraction rate can be as high as 8.57 mg / g. At the same time, the color value of the extracted chlorophyll is above 60, the chlorophyll content of the crude paste is above 50%, and there are fewer impurities. Traditional acetone extraction of silkworm feces is prone to acetone residue problems.

[0070] Even if the crude paste obtained by the method provided in Comparative Example 1 is subjected to desolventizing treatment according to the method provided in Comparative Example 2, the acetone content in the obtained product is still relatively high, and the effect of no acetone residue in the product obtained by the method provided in this application cannot be achieved.

[0071] When extracting chlorophyll from alfalfa using the method provided in this application, plant protein and chlorophyll can be separated. The resulting brownish-yellow extract, after concentration, is tested for the presence of some water-soluble protein. After precipitation with acetone, it can be collected and recycled. However, the methods of Comparative Examples 1 and 2 fail to produce an extract containing only water-soluble protein, and thus fail to separate chlorophyll from water-soluble protein. This requires subsequent refinement, increasing production costs.

[0072] The magnesium in the chlorophyll molecule is easily replaced under acidic conditions and heating, turning into pheophytin, which affects the activity of the resulting product.

[0073] The magnesium content in the obtained chlorophyll is directly related to the quality of the obtained chlorophyll. When the magnesium chlorophyll content is higher, the product quality is better. The magnesium content in the chlorophyll crude paste obtained in Example 1 and Comparative Example 3 was detected using an atomic absorption spectrometer AA-6300. The results are shown in the following table:

[0074] extract Magnesium content mg / g Comparative Example 3 6.02 Example 1 12.46

[0075] As can be seen from the above table, the extract obtained by the method provided by this application has a high magnesium content, which can better avoid magnesium denaturation and effectively improve the quality of the obtained product.

[0076] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting chlorophyll from alfalfa, characterized in that: The following steps are involved: The alfalfa is dried to a moisture content of less than 20% and then crushed to 80 meshes to obtain grass powder; the grass powder is pressed and rehydrated, and then percolation extraction is performed using alcohol as a solvent to obtain a brown-yellow extract and a green extract; the obtained green extract is concentrated under reduced pressure at 60°C to obtain a chlorophyll crude paste, and the color value of the obtained chlorophyll crude paste is above 60; the brown-yellow extract is concentrated under reduced pressure at 60°C to obtain a water-soluble protein crude paste; The rehydration operation is as follows: adding sodium carbonate solution to the compressed grass cake or grass ball and soaking it for 1 hour. The pH value of the solution during the rehydration process is alkaline.

2. The method for extracting chlorophyll from alfalfa according to claim 1, characterized in that: The drying process of alfalfa is to dry it in the sun for 2 days or to dry it at 80℃.

3. The method for extracting chlorophyll from alfalfa according to claim 1, characterized in that: The amount of sodium carbonate added to the sodium carbonate solution is 1-2% of the mass of the grass powder to be processed, and the mass of the aqueous solution used is 3 times the mass of the grass powder to be processed.

4. The method for extracting chlorophyll from alfalfa according to claim 1, characterized in that: The percolation extraction comprises the following steps: releasing the soaking liquid and simultaneously adding 80° alcohol into the extraction device during the release process, and the flow rate of the alcohol is the same as the flow rate of the soaking liquid.

5. The method for extracting chlorophyll from alfalfa according to claim 4, characterized in that: The flow rates of the immersion solution and alcohol are both 0.8 L / h.

6. The method for extracting chlorophyll from alfalfa according to claim 1, characterized in that: During the discharge process, the extract in the first 3-4 times amount is brown-yellow extract; during the last 6-7 times amount of extract is chlorophyll.

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