Method for producing extract containing natural chlorophyll

Abstract

To provide a method for producing an extract containing natural chlorophyll with a simple operation.SOLUTION: A method for producing an extract containing chlorophyll comprises a step of performing an extraction process on a plant body or alga containing chlorophyll by using an aqueous solution containing a compound represented by general formula (1) as an extractant.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing an extract containing natural chlorophyll by a simple operation, and a method for producing solid natural chlorophyll by a simple operation. [Background technology]

[0002] Chlorophyll is a green pigment found in plant leaves, algae, etc., and is also called chlorophyll. Chlorophyll is known to have effects such as anti-aging, cholesterol lowering, anti-allergy, metabolism promotion, dieting, rough skin improvement, deodorizing, intestinal environment improvement, excretion of harmful minerals from the body, hematopoiesis promotion, and cancer prevention, and is attracting attention as an ingredient for foods, cosmetics, medicines, etc.

[0003] Chlorophyll is broadly divided into artificial and natural types. Artificial chlorophyll is easy to produce because the central metal is replaced with copper or iron and it is extracted and purified using alkali or acid, but the side chains that should be there are cut during the extraction and purification process, and the functions that chlorophyll originally has are weakened or lost. On the other hand, natural chlorophyll maintains the structure that exists in nature, so it is more functional than artificial chlorophyll, but because advanced technology is required for extraction and purification, industrial production is difficult and the amount of distribution is small.

[0004] Therefore, various techniques for efficiently extracting natural chlorophyll have been studied. For example, Patent Document 1 describes that chlorophyll a can be efficiently produced by treating chlorella with 80 to 100% ethanol, extracting chlorophyll a, and then adding dioxane to the resulting extract to induce precipitation of chlorophyll a. However, the method of Patent Document 1 has the disadvantage that it uses high concentration ethanol and requires the use of dioxane to recover chlorophyll, making it an environmentally friendly technology. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special table 2010-509207 publication Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a method for producing an extract containing natural chlorophyll by a simple operation. Another object of the present invention is to provide a method for producing solid natural chlorophyll by a simple operation. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems and have found that natural chlorophyll can be efficiently extracted by using an aqueous solution containing a compound with a specific structure as an extraction solvent to extract plants or algae containing chlorophyll. Furthermore, the present inventors have found that natural chlorophyll can be precipitated by diluting the extract obtained by the above method with water, and can be easily recovered as solid natural chlorophyll. The present invention was completed based on these findings and through further research.

[0008] That is, the present invention provides the following aspects. Item 1. A method for producing an extract containing chlorophyll, comprising the step of extracting a plant or alga containing chlorophyll using an aqueous solution containing a compound represented by general formula (1) as an extraction solvent. [ka] [In the general formula (1), R1, R2, and R3 each represent the same or different alkyl group, and the total number of carbon atoms in the alkyl groups of R1, R2, and R3 is 3 to 15, R4 is an alkylene group having 1 to 5 carbon atoms, and Q - is the group -COO - or group -SO3 - It is.] Item 2. The method according to item 1, wherein in general formula (1), R1, R2, and R3 are alkyl groups having 4 or 5 carbon atoms, and R4 is an alkylene group having 1 to 3 carbon atoms. Item 3. The method according to item 1 or 2, wherein the concentration of the compound represented by general formula (1) in the aqueous solution is 0.3 to 10.0 M. Item 4. A chlorophyll-containing extract obtained by extracting a plant part or algae containing chlorophyll using an aqueous solution containing a compound represented by general formula (1) as an extraction solvent. [ka] [In the general formula (1), R1, R2, and R3 each represent the same or different alkyl group, and the total number of carbon atoms in the alkyl groups of R1, R2, and R3 is 3 to 15, R4 is an alkylene group having 1 to 5 carbon atoms, and Q - is the group -COO - or group -SO3 - It is.] Item 5. An extraction solvent for extracting chlorophyll from plant parts or algae, comprising an aqueous solution containing carboxybetaine represented by general formula (1). [ka] [In the general formula (1), R1, R2, and R3 each represent the same or different alkyl group, and the total number of carbon atoms in the alkyl groups of R1, R2, and R3 is 3 to 15, R4 is an alkylene group having 1 to 5 carbon atoms, and Q - is the group -COO - or group -SO3 - It is.] Item 6. A method for producing solid chlorophyll, comprising the steps of: A step of diluting the chlorophyll-containing extract according to item 1 with water to cause chlorophyll precipitation; The method further comprises the step of recovering the precipitate. Item 7. The method according to Item 6, wherein the extract is diluted with water to adjust the concentration of the compound represented by general formula (1) contained in the extract to 1.3 M or less. Effect of the Invention

[0009] According to the present invention, chlorophyll can be efficiently extracted by using an aqueous solution containing a compound represented by general formula (1) as an extraction solvent and extracting a plant or alga containing chlorophyll. Furthermore, since chlorophyll can be precipitated by diluting the extract obtained using the extraction solvent with water, solid chlorophyll can be recovered by a simple operation. The method for producing an extract containing chlorophyll of the present invention can extract and recover poorly water-soluble chlorophyll without using ethanol, etc., and therefore can be said to be an extraction technique for chlorophyll with low environmental impact. [Brief description of the drawings]

[0010] [Figure 1] This is the UV spectrum of an extract obtained by extracting chlorophyll from dried powder of Moringa leaves using a 2.0 M aqueous solution of carboxybetaine 5 (a compound represented by the general formula (1) in which R1 to R3 are linear pentyl groups, R4 is a methylene group, and Q- is a -COO- group). In FIG. 1, the absorbance on the vertical axis is the value obtained by multiplying the measured absorbance value by the dilution ratio of the extract used for the measurement. [Diagram 2] Chlorophyll was extracted from dried powder of moringa leaves using a 0 to 2.4 M aqueous solution of carboxybetaine 5, and the absorbance at 665 nm derived from the chlorophyll contained in the extract was measured. [Diagram 3] Chlorophyll was extracted from dried powder of moringa leaves using a 0 to 2.0 M aqueous solution of carboxybetaine 4, and the absorbance at 665 nm derived from the chlorophyll contained in the extract was measured. [Figure 4] Chlorophyll was extracted from dried powder of moringa leaves at 4 to 80°C using an aqueous solution of carboxybetaine 5, and the absorbance at 665 nm derived from the chlorophyll contained in the extract was measured. [Diagram 5]Chlorophyll was extracted from dried moringa leaf powder using an aqueous solution of carboxybetaine 5 for 10 minutes to 3 hours, and the absorbance at 665 nm derived from the chlorophyll contained in the extract was measured. [Figure 6] Chlorophyll was extracted from 1 to 40 mg of dried moringa leaf powder using 300 μL of carboxybetaine 5 aqueous solution, and the absorbance at 665 nm derived from the chlorophyll contained in the extract was measured. [Figure 7] Chlorophyll was extracted from dried powder of moringa leaves using ethanol, acetone, ethyl acetate, or carboxybetaine 5, and the absorbance at 665 nm derived from the chlorophyll contained in the extract was measured. [Figure 8] The extract extracted from dried powder of moringa leaves using a 2.0 M aqueous solution of carboxybetaine 5 was diluted 1.5 to 50 times with distilled water, and the amount of precipitate formed was measured. [Figure 9] The extract extracted from dried powder of Moringa leaves using a 2.0 M aqueous solution of carboxybetaine 4 (a compound in which R1 to R3 are n-butyl groups, R4 is a methylene group, and Q- is a -COO- group in general formula (1)) was diluted 1.5 to 12.5 times with distilled water, and the amount of precipitate formed was measured. [Figure 10] The extract extracted from dried powder of moringa leaves using a 2.0 M aqueous solution of carboxybetaine 5 was diluted 4-fold with distilled water and allowed to stand for 1 to 26 hours, after which the amount of precipitate formed was measured. [Figure 11] The extract extracted from dried powder of Chlorella using a 2.0 M aqueous solution of carboxybetaine 5 was diluted 1.5 to 12.5 times with distilled water, and the amount of precipitate formed was measured. [Figure 12] Chlorophyll was extracted from dried powder of moringa leaves using a 0-2.0 M aqueous solution of sulfobetaine 4 (a compound represented by general formula (1) in which R1 to R3 are n-butyl groups, R4 is a propylene group, and Q- is a -SO3- group), and the absorbance at 665 nm derived from the chlorophyll contained in the extract was measured. [Figure 13] Chlorophyll was extracted from dried powder of moringa leaves using a 0-2.0 M aqueous solution of sulfobetaine 5 (a compound represented by general formula (1) in which R1 to R3 are linear pentyl groups, R4 is a propylene group, and Q- is a -SO3- group), and the absorbance at 665 nm derived from the chlorophyll contained in the extract was measured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1. Method for producing extract containing chlorophyll In the production method of the present invention, an aqueous solution containing the compound represented by the general formula (1) is used as an extraction solvent to extract a plant or algae containing chlorophyll, thereby obtaining an extract containing chlorophyll. The production method of the present invention will be described in detail below.

[0012] [Extraction solvent] In the production method of the present invention, an aqueous solution containing the compound represented by formula (1) is used as an extraction solvent. That is, in the production method of the present invention, an extraction solvent containing the compound represented by formula (1) as a solute and water as a solvent is used.

[0013] The structure of the compound represented by general formula (1) is as follows. [ka]

[0014] In the general formula (1), R1, R2, and R3 each represent the same or different alkyl group, and the total number of carbon atoms in the alkyl groups of R1, R2, and R3 is 3 to 15. In the present invention, the "total number of carbon atoms in the alkyl groups of R1, R2, and R3" refers to the total value of the number of carbon atoms in the alkyl group of R1, the number of carbon atoms in the alkyl group of R2, and the number of carbon atoms in the alkyl group of R3. By using a compound in which the total number of carbon atoms in the alkyl groups of R1, R2, and R3 falls within the above range, it becomes possible to efficiently extract chlorophyll. From the viewpoint of more efficient extraction of chlorophyll, the total number of carbon atoms in the alkyl groups of R1, R2, and R3 is preferably 10 to 15, more preferably 11 to 15, even more preferably 12 to 15, even more preferably 12 or 15, and particularly preferably 15.

[0015] The number of carbon atoms in each of the alkyl groups R1, R2, and R3 is not particularly limited as long as the total number of carbon atoms falls within the above-mentioned range, and may be, for example, 1 to 8, preferably 2 to 7, more preferably 3 to 6, even more preferably 3 to 5, still more preferably 4 or 5, and particularly preferably 5.

[0016] When the alkyl group of R1, R2, and R3 has 3 or more carbon atoms, the alkyl group may be either linear or branched, and is preferably linear.

[0017] In general formula (1), R4 is an alkylene group having 1 to 5 carbon atoms. R4 is preferably an alkylene group having 1 to 4 carbon atoms, and more preferably a methylene group (-CH2-), an ethylene group (-CH2-CH2-), or a propylene group (-CH2-CH2-CH2-).

[0018] In the general formula (1), Q - is the group -COO - (a group formed by removing a proton from a carboxy group), or the group -SO3 - (a group formed by removing a proton from a sulfo group).

[0019] A preferred example of the compound represented by the general formula (1) is a compound represented by the general formula (1), in which R1, R2, and R3 are the same or different and each is an alkyl group having 3 to 5 carbon atoms, R4 is an alkylene group having 1 to 3 carbon atoms, and Q - Ga-ki COO - More preferably, in the general formula (1), R1, R2, and R3 are the same or different and each is an alkyl group having 4 or 5 carbon atoms (preferably an n-butyl group or a linear pentyl group), R4 is a methylene group, and Q - Ga-ki COO - More preferably, in the general formula (1), R1, R2, and R3 are linear pentyl groups, R4 is a methylene group, and Q - Ga-ki COO - Examples of such compounds include those in which:

[0020] Other preferable examples of the compound represented by the general formula (1) include those in which, in the general formula (1), R1, R2, and R3 are the same or different and each is an alkyl group having 3 to 5 carbon atoms, R4 is an alkylene group having 1 to 3 carbon atoms, and Q - Base -SO3 - More preferably, in the general formula (1), R1, R2, and R3 are the same or different and each is an alkyl group having 4 or 5 carbon atoms (preferably an n-butyl group or a linear pentyl group), R4 is a propylene group, and Q - Base -SO3 - More preferably, in the general formula (1), R1, R2, and R3 are linear pentyl groups, R4 is a propylene group, and Q - Base -SO3 - Examples of such compounds include those in which:

[0021] In the extraction solvent, the compound represented by the general formula (1) may contain one type of structure alone, or may contain two or more types of structures in combination.

[0022] A method for producing the compound represented by the general formula (1) is publicly known, for example, in JP-A-2009-96766, and can be derived from a publicly known organic synthesis method.

[0023] The concentration of the compound represented by general formula (1) in the aqueous solution used as the extraction solvent may be appropriately set depending on the structure of the compound, the type of plant or algae to be subjected to the extraction treatment, etc., and may be, for example, 0.3 M or more, preferably 0.3 to 10 M. More specifically, in general formula (1), R1, R2, and R3 are linear pentyl groups, R4 is an alkylene group having 1 to 3 carbon atoms, and Q - Ga-ki COO - or group -SO3 - In the case of using a compound represented by the formula (1), the concentration of the compound in the aqueous solution used as the extraction solvent is 0.3 M or more, preferably 0.3 to 3 M, more preferably 0.5 to 2.4 M or 0.5 to 3.0 M. In addition, in the formula (1), R1, R2, and R3 are n-butyl groups, R4 is an alkylene group having 1 to 3 carbon atoms, and Q - Ga-ki COO - or group -SO3 - When a compound represented by the formula (I) is used, the concentration of the compound in the aqueous solution used as the extraction solvent is 0.5M or more, preferably 0.7 to 10M, and more preferably 1.0 to 2.0M.

[0024] The extraction solvent may be any aqueous solution containing a water solvent, and may contain other solvents, such as water-soluble organic solvents such as methanol, ethanol, etc., as long as the effects of the present invention are not lost. In addition, the extraction solvent may contain additives, such as surfactants, as long as the effects of the present invention are not lost.

[0025] [Extraction raw material] In the production method of the present invention, a plant body and / or algae containing chlorophyll is used as the extraction raw material.

[0026] The type of plant used as the extraction raw material is not particularly limited as long as it contains chlorophyll, but examples include moringa leaves, shiso leaves, olive leaves, tea leaves, spinach, comfrey, alfalfa, kale, young barley leaves, young wheat leaves, angelica leaves, young mulberry leaves, komatsuna, asparagus, green peppers, kidney beans, chrysanthemum, bok choy, ascetic greens, broccoli, radish leaves, mulberry leaves, beet leaves, soybean leaves, corn leaves, ginkgo leaves, all-purpose onions, hijiki, shishito peppers, watercress, peas, green vegetables, kale, mustard greens, Japanese parsley, shepherd's purse, mugwort, Chinese chives, Nozawana, garlic sprouts, parsley, scallions, mulukhiyah, mitsuba, Brussels sprouts, basil, etc. These plants may be used alone or in combination of two or more. In addition, the plant body used as the extraction raw material is preferably chopped or powdered in order to increase the efficiency of chlorophyll extraction.

[0027] The type of algae used as the extraction raw material is not particularly limited as long as it contains chlorophyll, and examples thereof include green algae, red algae, brown algae, cryptophytes, raphidophytes, dinoflagellates, euglena plants, chlorarachniophytes, glaucophytes, diatoms, and cyanobacteria. These algae may be used alone or in combination of two or more. In addition, the algae used as the extraction raw material is preferably chopped or powdered to increase the efficiency of chlorophyll extraction.

[0028] The plant body and / or algae used as the extraction raw material may be one that has been subjected to a drying treatment, or may be one that has not been dried.

[0029] [Extraction process] In the production method of the present invention, the extraction treatment may be performed by immersing the raw material in the extraction solvent. During the extraction treatment, stirring may be performed as necessary.

[0030] In the extraction treatment, the amount of the extraction raw material added to the extraction solvent is not particularly limited, but the amount of the plant body and / or algae in the extraction solvent is 0.001 to 200 mg / mL, preferably 0.1 to 200 mg / mL, and more preferably 1 to 150 mg / mL, calculated as dry weight.

[0031] The temperature during the extraction treatment is not particularly limited, but may be, for example, 0 to 100°C, and preferably 4 to 80°C.

[0032] The extraction treatment time is not particularly limited, but may be, for example, 1 minute to 72 hours, preferably 1 to 24 hours, and more preferably 3 to 17 hours.

[0033] The solid content is removed from the suspension after the extraction treatment to obtain an extract. The removal of the solid content can be performed by a known solid-liquid separation method such as filtering or centrifugation. The obtained extract contains chlorophyll eluted from the plant body and / or algae and the compound represented by general formula (1).

[0034] The obtained extract may be used as a chlorophyll-containing liquid in various products such as foods, cosmetics, and pharmaceuticals, either as is or after concentration, or the obtained extract may be subjected to the "Method for producing solid chlorophyll" described below to recover solid chlorophyll.

[0035] 2. How to make solid chlorophyll In the manufacturing method of the present invention, a step of diluting the extract obtained in the extraction treatment with water to cause chlorophyll precipitation and a step of recovering the precipitate are carried out, thereby obtaining solid chlorophyll. Even when water is added to the extract, the compound represented by general formula (1) remains dissolved and does not precipitate, so that the compound is separated from the recovered precipitate (solid chlorophyll).

[0036] The water used to dilute the extract may be distilled water, purified water, tap water, or the like, or may be a buffer solution or the like.

[0037] The amount of water added required to precipitate chlorophyll from the extract cannot be uniformly determined because it varies depending on the structure and concentration of the compound represented by general formula (1) contained in the extract, the type of plant and / or algae used as the extraction raw material, etc., but may be set so that the compound represented by general formula (1) contained in the extract is diluted to 1.3 M or less, preferably 1.0 M or less, by adding water. More specifically, in general formula (1), R1, R2, and R3 are linear pentyl groups, R4 is an alkylene group having 1 to 3 carbon atoms, and Q - Ga-ki COO - or group -SO3 - In the case of using a compound represented by the formula (1), the compound contained in the extract may be diluted to 1.3 M or less, preferably 1.0 M or less, more preferably 0.7 M or less by adding water. - Ga-ki COO - or group -SO3 - When a compound represented by the formula (I) is used, the concentration of the compound contained in the extract may be set to be 0.5 M or less, preferably 0.45 M or less, and more preferably 0.4 M or less.

[0038] The temperature conditions when water is added to the extract are not particularly limited and may be, for example, 4 to 80° C., preferably 10 to 40° C. Furthermore, the addition of water to the extract causes precipitation of chlorophyll, and in order to increase the amount of chlorophyll precipitate, it is desirable to add water to the extract, hold the mixture for 1 minute to 72 hours, preferably 1 to 48 hours, more preferably 6 to 25 hours, and then recover the precipitate.

[0039] The extract is diluted with water, and the resulting chlorophyll precipitate is collected to obtain solid chlorophyll. The precipitate can be collected by a known solid-liquid separation method such as filtering or centrifugation. The collected precipitate (solid chlorophyll) may be subjected to a washing process as necessary. The collected precipitate (solid chlorophyll) may be subjected to a drying process as necessary to obtain a dried chlorophyll. The obtained solid chlorophyll can be used in various products such as food, cosmetics, and medicines. EXAMPLES

[0040] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0041] 1. Test Materials 1-1. Synthesis of the compound represented by formula (1) Carboxybetaines 4 and 5 shown below were synthesized with reference to the method described in JP 2009-96766 A. Sulfobetaines 4 and 5 shown below were synthesized by known methods. Carboxybetaine 4 is a compound represented by the general formula (1) in which R1 to R3 are n-butyl groups, R4 is a methylene group, and Q - Ga-ki COO - Carboxybetaine 5 is a compound represented by the general formula (1), in which R1 to R3 are linear pentyl groups, R4 is a methylene group, and Q - Ga-ki COO - Sulfobetaine 4 is a compound represented by the general formula (1), in which R1 to R3 are n-butyl groups, R4 is a propylene group, and Q is a propylene group. - Base -SO3 - Sulfobetaine 5 is a compound represented by the general formula (1), in which R1 to R3 are linear pentyl groups, R4 is a propylene group, and Q - Base -SO3 - It is a compound wherein [ka]

[0042] 1-2. Preparation of extraction materials (1) Dried moringa leaf powder The product name "Moringa Leaf Dried Powder" (Mitoyo Superfood Production Association) was obtained and used as the dried powder of moringa leaves.

[0043] (2) Dried powder and wet Chlorella Wet chlorella (undried chlorella) was collected from the chlorella culture solution. The undried chlorella was placed in a petri dish and heated at 80°C for 1 hour to dry it. It was then crushed in a Wonder Blender and passed through a 350μm sieve to collect the particles that passed through and used as dried chlorella powder.

[0044] (3) Dried powder and wet shiso leaves Fresh shiso leaves were heated at 80°C for 1 hour and dried. Next, they were crushed in a Wonder Blender and sieved through a 350μm sieve, and the pieces that passed through were collected and used as dried shiso powder. Fresh shiso leaves were also cut into 2mm cubes with scissors without drying and used as undried shiso leaves.

[0045] (4) Dried and wet olive leaves Fresh olive leaves were heated at 80°C for 2 hours and dried. Then, after crushing with a Wonder Blender, they were sieved through a 350μm sieve, and the pieces that passed through were collected and used as dried olive powder. Fresh olive leaves were also cut into 2mm cubes with scissors under the same conditions as above without drying, and used as undried olive leaves.

[0046] (5) Measurement of moisture content of extraction raw materials The moisture content of the extraction raw material was measured by the following method. First, a certain amount of sample was placed in an aluminum petri dish, and the initial weight of the petri dish containing the sample was measured. Next, the petri dish containing the sample was placed in a vacuum heating dryer (EYELA, VOS-201 SD), heated to 70°C, and left to stand at a constant temperature while creating a vacuum with a diaphragm vacuum pump. The petri dish was taken out at regular intervals and the weight was measured, and this was repeated until there was no change in weight. The moisture content was calculated from the final weight and the initial weight. The moisture content measurement results are shown in Table 1.

[0047] [Table 1]

[0048] 2. Carboxybetaine (Q in general formula (1) - Ga-ki COO - Extraction and recovery of chlorophyll using the compound 2-1. Test Example 1 (Extraction of chlorophyll using Carboxybetaine 5) Chlorophyll was extracted from the plant body using an extraction solvent (carboxybetaine 5 aqueous solution) prepared by dissolving carboxybetaine 5 in purified water to a concentration of 2.0 M. Dried powder of moringa leaves was used as the plant body containing chlorophyll.

[0049] 30 mg of dried powder of moringa leaves was placed in a 1.5 mL plastic tube, and 300 μL of betaine 5 aqueous solution was added. The powder was mixed and stirred well using a vortex mixer so that it was uniformly dispersed in the solution, and the extract attached to the wall of the tube was collected at the bottom by centrifuging with a small tabletop centrifuge. The tube was then incubated at room temperature (20 to 25 ° C) for 3 hours. The extract suspension containing the moringa plant body was then centrifuged (16,000 g, 5 minutes, room temperature, Micro-12 (Greiner)), all solids were collected at the bottom, and the supernatant (extract) was taken out and the UV spectrum was measured. The UV spectrum was measured by diluting the extract with carboxybetaine 5 aqueous solution.

[0050] The obtained UV spectrum is shown in Figure 1. As a result, an absorption peak at 665 nm specific to chlorophyll was confirmed in the extract. In other words, it was made clear that chlorophyll can be extracted by soaking moringa leaves in an aqueous solution of carboxybetaine 5.

[0051] 2-2. Test Example 2 (Chlorophyll extraction amount when the concentration of Carboxybetaine 5 is changed) In order to confirm whether the extraction of chlorophyll confirmed in Test Example 1 was due to the addition of carboxybetaine 5 to the aqueous solution, the concentration of carboxybetaine 5 was changed in the range of 0 to 2.4 M, and chlorophyll was extracted from dried moringa powder. The specific experimental conditions were the same as those in Test Example 1, except that the concentration of carboxybetaine 5 was changed.

[0052] The results are shown in Figure 2. Figure 2 plots the absorbance at 665 nm derived from chlorophyll contained in the extract after extraction treatment against the concentration of carboxybetaine 5. When no carboxybetaine 5 was added (0 M), no peak at 665 nm derived from chlorophyll was observed, but when carboxybetaine 5 was added, a peak at 665 nm was detected. In other words, this result confirmed that carboxybetaine 5 is involved in the extraction of chlorophyll from the plant body.

[0053] It was also confirmed that when the concentration of carboxybetaine 5 was in the range of 0.5 to 1.7 M, the absorbance at 665 nm increased in a concentration-dependent manner with respect to carboxybetaine 5, and when the concentration exceeded 1.7 M, the absorbance at 665 nm decreased slightly.

[0054] 2-3. Test Example 3 (Difference in the amount of chlorophyll extracted due to differences in the structure of carboxybetaine) In Test Examples 1 and 2, it was confirmed that chlorophyll can be extracted by using an aqueous solution in which carboxybetaine 5 is dissolved. Therefore, in this Example, an extraction experiment was carried out using carboxybetaine having a structure different from that of carboxybetaine 5. The specific experimental conditions were the same as those in Test Example 1, except that carboxybetaine 4 (2.0 M) was used.

[0055] The absorbance at 665 nm derived from chlorophyll in the obtained UV spectrum is shown in Table 2. Note that Table 2 also shows the results of carboxybetaine 5 (2.0 M) in Example 1. As a result, it was confirmed that chlorophyll can be extracted from the dried powder of moringa even when carboxybetaine 4 is used. However, when carboxybetaine 4 was used, the absorbance was about one-fourth that of carboxybetaine 5. In other words, it was shown that structures such as carboxybetaine 4 and 5 are necessary to extract chlorophyll, and that the most suitable carboxybetaine structure among them is carboxybetaine 5.

[0056] In addition, since the amount of carboxybetaine dissolved in an aqueous solution depends on the molecular weight and chemical structure of the carboxybetaine, even in the case of a carboxybetaine having fewer carbon atoms in the alkyl groups R1 to R3 than in carboxybetaine 5, it is possible to improve the extraction of chlorophyll by increasing the amount of carboxybetaine dissolved and increasing the concentration.

[0057] [Table 2]

[0058] 2-4. Test Example 4 (Chlorophyll extraction amount when the concentration of Carboxybetaine 4 is changed) Since it was confirmed in Test Example 3 that chlorophyll can be extracted even when carboxybetaine 4 is used, in this Test Example, chlorophyll extraction processing was performed from dried moringa powder by changing the concentration of carboxybetaine 4 from 0 to 2.0 M. The specific experimental conditions were the same as in Test Example 3, except that the concentration of carboxybetaine 4 was changed.

[0059] The results are shown in Figure 3. In Figure 3, the absorbance at 665 nm derived from chlorophyll contained in the extract after extraction treatment is plotted against the concentration of carboxybetaine 4. As a result, it was confirmed that carboxybetaine 4 increases the amount of chlorophyll extracted in a concentration-dependent manner at concentrations of 0.5 to 2.0 M.

[0060] 2-5. Test Example 5 (Effect of extraction temperature on the amount of chlorophyll extracted by Carboxybetaine 5) In Test Example 1, chlorophyll extraction was performed at room temperature (20-25°C). In this Test Example, the extraction temperature was changed from 4 to 80°C to examine the effect of the extraction temperature on the amount of chlorophyll extracted. The specific experimental conditions were the same as Test Example 1, except for the change in the extraction temperature.

[0061] The results are shown in Figure 4. Figure 4 plots the 665 nm absorbance derived from chlorophyll contained in the extract after extraction against the extraction temperature. No significant difference was observed in the 665 nm absorbance derived from chlorophyll between 4°C and 80°C. In other words, the effect of the extraction temperature was small, demonstrating that chlorophyll can be extracted using an aqueous solution of carboxybetaine 5 over a wide range of extraction temperatures.

[0062] 2-6. Test Example 6 (Effect of extraction time on the amount of chlorophyll extracted by Carboxybetaine 5) In Test Example 1, the extraction process was carried out for 3 hours. In this example, the extraction time was changed from 10 minutes to 24 hours, and the effect of the extraction time on the amount of chlorophyll extracted was examined. The specific experimental conditions were the same as those in Test Example 1, except for the change in the extraction time.

[0063] The results are shown in Figure 5. Figure 5 plots the absorbance at 665 nm derived from the chlorophyll contained in the extract after extraction against the extraction time. The absorbance at 665 nm increased between 10 minutes and 3 hours of extraction time, and remained constant for 3 hours. In other words, this result confirmed that chlorophyll can be sufficiently extracted if the extraction is performed for 3 hours or more.

[0064] 2-7. Test Example 7 (Relationship between the amount of dried powder of moringa leaves and the amount of chlorophyll extracted by betaine 5) In Test Example 1, the amount of dry powder of Moringa leaves was fixed at 30 mg and an extraction experiment was performed. In this example, the amount of dry powder of Moringa leaves was changed from 1 to 40 mg, and the effect of the amount of dry powder of Moringa leaves on the amount of chlorophyll extracted was examined. The specific experimental conditions were the same as Test Example 1, except that the amount of dry powder of Moringa leaves was changed.

[0065] The results are shown in Figure 6. Figure 6 plots the absorbance at 665 nm derived from chlorophyll contained in the extract after extraction against the amount of dried powder of moringa leaves. From 1 to 40 mg, the absorbance at 665 nm derived from the extracted chlorophyll increases proportionally to the amount of powder, confirming that the amount of chlorophyll extracted increases as the amount of powder increases.

[0066] 2-8. Reference Test Example 1 (Extraction of chlorophyll using organic solvents) In order to compare the extraction of chlorophyll using carboxybetaine 5 with the extraction using existing organic solvents, chlorophyll was extracted from dry powder of moringa leaves using ethanol, acetone, or ethyl acetate instead of aqueous solution of betaine 5 (2M). The specific experimental conditions were the same as those in Test Example 1, except that the extraction solvent was changed and the amount of dry powder of moringa leaves was changed to 25 mg.

[0067] The results are shown in Figure 7. Figure 7 shows the absorbance at 665 nm of each of the obtained extracts. As a result, the aqueous solution of carboxybetaine 5 (2M) extracted more chlorophyll than acetone or ethyl acetate, and the amount of chlorophyll extracted was equivalent to that of ethanol. Since the aqueous solution of carboxybetaine 5 is a water solvent, it is easier to handle and has a smaller environmental impact than organic solvents such as ethanol, making it more advantageous than existing organic solvents as an extraction solvent used in chlorophyll extraction processing.

[0068] 2-9. Test Example 8 (Recovery of chlorophyll from extract) As shown in Test Examples 1-3 and 5-7, it was confirmed that the aqueous solution of carboxybetaine 5 is an excellent extraction solvent for the extraction of chlorophyll. In this test example, the recovery operation from the extract was examined. Test Example 2 shows that the extraction of chlorophyll varies depending on the concentration of carboxybetaine 5. Therefore, a method was examined in which the chlorophyll extract obtained by the extraction treatment using an aqueous solution of carboxybetaine 5 is diluted with water to reduce the concentration of carboxybetaine 5 to a level that is not suitable for dissolving chlorophyll, and the chlorophyll is recovered as a precipitate. This method does not require a pressure reducing device such as vacuum distillation, so it can be said to be a beneficial method in that it can utilize existing equipment for industrial use.

[0069] First, an extract containing chlorophyll was obtained under the same conditions as in Test Example 1. 20 μL of the obtained extract was mixed with 60 μL of distilled water (dilution ratio 4 times, final concentration of carboxybetaine 5 was 0.5 M) and incubated at room temperature (20 to 25 ° C) for 1 hour. Next, the mixture was centrifuged (16,000 g, 5 minutes, room temperature, Greiner, Micro-12), and the resulting precipitate was collected at the bottom. The supernatant was removed by decantation, 200 μL of distilled water was added, and the mixture was stirred with a vortex mixer to wash off the carboxybetaine 5 adhering to the precipitate. This washing operation was repeated three times, and the precipitate was freeze-dried.

[0070] The yield of the resulting precipitate (lyophilized material) was measured to be 0.13 mg (0.4% by weight relative to the dry powder of moringa leaves used for extraction). Based on the absorbance value at 665 nm of the extract and the absorbance value of the supernatant of a mixture obtained by diluting the extract four-fold, it was estimated that 85% of the extracted chlorophyll was recovered as precipitate by diluting with distilled water. These results demonstrated that solid chlorophyll can be efficiently recovered by the simple procedure of diluting the chlorophyll extract obtained using carboxybetaine 5 with water.

[0071] 2-10. Test Example 9 (Effect of dilution ratio on recovery of chlorophyll from chlorophyll extract) In Test Example 8, it was shown that solid chlorophyll can be recovered by diluting an extract containing chlorophyll with water. Therefore, in this Test Example, the effect of the dilution ratio when diluting an extract containing chlorophyll on the recovery rate of chlorophyll was examined. The specific experimental conditions were the same as Test Example 7, except that the amount of distilled water added was changed to 10 to 980 μL (dilution ratio 1.5 to 50 times, final concentration of carboxybetaine 5 0.04 to 1.3 M).

[0072] The results are shown in Figure 8. Figure 8 shows the correlation between the dilution ratio and the weight of the resulting precipitate (lyophilized product). It was found that the maximum amount of precipitate was obtained when the extract containing chlorophyll was diluted 10 times with distilled water (final concentration of carboxybetaine 5: 0.2 M). It was thought that the amount of precipitate would increase as the dilution ratio increased because the solubility of chlorophyll decreased, but a tendency for the amount of precipitate to decrease was observed at dilution ratios of more than 10. This is presumably because when the dilution ratio is too high, aggregation (precipitation) of chlorophyll in the solution becomes unfavorable.

[0073] 2-11. Test Example 10 (Effect of dilution ratio on recovery of chlorophyll from betaine 4 aqueous solution (extraction solution)) As shown in Test Example 3, it has been confirmed that chlorophyll can be extracted using an aqueous solution containing carboxybetaine 4. In this Test Example, it was verified whether the chlorophyll precipitate could be recovered by diluting the extract obtained using an aqueous solution containing carboxybetaine 4 with distilled water. The specific experimental conditions were the same as those in Test Example 9, except that carboxybetaine 5 was replaced with carboxybetaine 4.

[0074] The results are shown in Figure 9. Figure 9 shows the correlation between the dilution ratio and the weight of the precipitate (lyophilized product) obtained. When the extract obtained using carboxybetaine 4 was diluted with distilled water, a bell-shaped plot was obtained, similar to the case of carboxybetaine 5 (Figure 8), indicating that there is an optimal dilution ratio. However, in the case of carboxybetaine 4, unlike carboxybetaine 5, the optimal dilution ratio of the extract was 7.5 times. From these results, it was found that when the extract obtained using carboxybetaine is diluted to recover chlorophyll precipitate, there is an optimal dilution ratio according to the structure of the carboxybetaine.

[0075] 2-12. Test Example 11 (Optimal Standing Time for Obtaining the Maximum Amount of Chlorophyll Precipitation after Dilution of Extract) In Test Example 8, it was shown that solid chlorophyll can be recovered by diluting an extract containing chlorophyll with water. Chlorophyll molecules aggregate in the diluted extract to cause precipitation, and this test example examined how long the extract should be left to stand to obtain the maximum amount of precipitation. Specifically, extraction was performed in the same manner as in Test Example 8, except that the time left to stand after dilution was changed from 1 to 26 hours.

[0076] The results are shown in Figure 10. The amount of precipitate increased over time until the standing time was 6 hours, but after 6 hours, almost no change in the amount of precipitate was observed. In other words, it was clear that the maximum amount of precipitate could be obtained by leaving the extract for 6 hours or more after dilution.

[0077] 2-13. Reference Test Example 2 (Recovery of chlorophyll by diluting the extract using an organic solvent) In Test Examples 8 to 11, it was confirmed that solid chlorophyll can be easily recovered by diluting the extract obtained using carboxybetaine 4 or 5 with distilled water. Therefore, it was verified whether solid chlorophyll can be recovered by diluting the extract obtained using ethanol or acetone as the extraction solvent with distilled water. The specific experimental conditions were the same as those of Test Example 8, except that the extract obtained using ethanol or acetone was used as the extraction solvent.

[0078] As a result, when the extract obtained using ethanol was diluted with distilled water, turbidity occurred, but no precipitate was obtained in an amount that could be weighed. Furthermore, when the extract obtained using acetone was diluted with distilled water, no turbidity was observed. In other words, it was found that the phenomenon of chlorophyll precipitation occurring when an extract containing chlorophyll is diluted with water is a unique phenomenon observed when an aqueous solution containing carboxybetaine 4 and 5 is used as the extraction solvent.

[0079] 2-14. Test Example 12 (Extraction of chlorophyll from other plants or algae) As described above, it was confirmed that chlorophyll can be extracted and recovered from dried powder of moringa leaves by using an aqueous solution of carboxybetaine 5 as an extraction solvent. We verified whether this extraction and recovery technology can be applied not only to moringa leaves but also to other plants or algae that contain chlorophyll.

[0080] First, the extraction process was performed under the same conditions as in Test Example 1, except that dried powder of chlorella, dried powder of olive leaves, or dried powder of shiso leaves was used instead of dried powder of moringa leaves. The results of measuring the absorbance at 665 nm of the obtained extract are shown in Table 3. As a result, it was confirmed that chlorophyll can be extracted from dried powder of chlorella, dried powder of olive leaves, and dried powder of shiso leaves by using an aqueous solution of carboxybetaine 5. In particular, when dried powder of chlorella and dried powder of shiso leaves were used, 2 to 4 times more chlorophyll could be extracted than dried powder of moringa leaves. On the other hand, when dried powder of olive leaves was used, the amount of chlorophyll extracted was about one-third compared to dried powder of moringa leaves. This is thought to be due to the difference in the amount of chlorophyll contained in the plant body or algae. From the above results, it was confirmed that chlorophyll can be extracted not only from moringa leaves but also from other plants or algae containing chlorophyll by using an aqueous solution of carboxybetaine 5.

[0081] [Table 3]

[0082] Next, using each of the extracts obtained above, chlorophyll precipitates were collected under the same conditions as in Test Example 8. As a result, the amount of precipitate (lyophilized material) that could be collected was 0.040 mg (dried powder of chlorella), 0.070 mg (dried powder of olive leaves), and 0.122 mg (dried powder of shiso leaves).

[0083] Although a large amount of chlorophyll was extracted from the extract obtained using dried powder of Chlorella, the amount of chlorophyll precipitate recovered was relatively small. This is thought to be due to the fact that the dilution ratio of the extract when causing precipitation was not optimal. Therefore, the optimal dilution ratio for causing precipitation was examined using the extract obtained using dried powder of Chlorella in the same manner as in Test Example 9. The results are shown in Figure 11. Figure 11 shows the correlation between the dilution ratio and the weight of the precipitate obtained. Unlike the results of Test Examples 8 and 9, the most chlorophyll precipitate was generated when the dilution ratio of the extract was 4 to 5 times. In other words, it was revealed that when the extract obtained using carboxybetaine is diluted to recover chlorophyll precipitate, there is an optimal dilution ratio depending not only on the structure of carboxybetaine but also on the type of plant body used.

[0084] 2-15. Test Example 13 (Effect of dryness of plant body on extraction of chlorophyll) In Test Example 12, dried powder was used as the plant body, but we verified whether chlorophyll can be extracted from undried raw materials. The specific experimental conditions were the same as those in Test Example 1, except that undried chlorella, undried olive, or undried shiso leaves were used as the plant body or algae.

[0085] The absorbance at 665 nm derived from the chlorophyll contained in each of the obtained extracts was measured, and the results are shown in Table 4. As a result, it was confirmed that chlorophyll could be extracted by the aqueous solution of Carboxybetaine 5 regardless of the undried matter used. In other words, it was confirmed that the extraction of chlorophyll by the aqueous solution of Carboxybetaine 5 is not affected by the properties (water content) of the tissue, and can be applied to plants or algae of any properties.

[0086] [Table 4]

[0087] 3. Sulfobetaine (Q in general formula (1) - -SO 3 - Chlorophyll extraction by Test Example 12 (Extraction of chlorophyll using sulfobetaine 4 and 5) In the above Test Examples 1 to 3, chlorophyll was successfully extracted using an aqueous solution containing carboxybetaine 4 or 5 dissolved therein. - Ga-ki COO - From group -SO3 - In order to confirm whether chlorophyll can be extracted even if the extraction solvent is changed to sulfobetaine 4 or 5, an extraction experiment was carried out using sulfobetaine 4 and 5. The extraction was carried out in the same manner as in Test Example 2, except that sulfobetaine 4 or 5 was used instead of carboxybetaine 4 or 5 dissolved in the aqueous solution.

[0088] The results of chlorophyll extraction using sulfobetaine 4 are shown in FIG. 12, and the results of chlorophyll extraction using sulfobetaine 5 are shown in FIG. 13. As a result, it was confirmed that chlorophyll can be efficiently extracted using sulfobetaine 4 and 5. Also, as in the case of carboxybetaine 4 and 5, sulfobetaine 5, in which R1, R2, and R3 in general formula (1) are linear pentyl groups, had a higher chlorophyll extraction efficiency than sulfobetaine 4. Also, when sulfobetaine 4 or 5 was used, chlorophyll could be extracted at a concentration of 0.5 M or more, and in general formula (1), Q - Group-COO - Not limited to, the group -SO3 - It was found that efficient extraction of chlorophyll was possible even with compounds that were too acidic.

[0089] Furthermore, the extract obtained using sulfobetaine 4 or 5 was diluted under the same experimental conditions as in Test Example 8, and the resulting precipitate was recovered. As a result, 0.157 mg of precipitate was recovered for sulfobetaine 4, and 0.584 mg for sulfobetaine 5. This indicates that the recovery procedure after extraction can be carried out in the same manner even when sulfobetaine is used.

Claims

1. A method for producing an extract containing chlorophyll, comprising the step of extracting a plant or alga containing chlorophyll using an aqueous solution containing a compound represented by general formula (1) as an extraction solvent. [Formula 1] [In the general formula (1), R 1 , R 2 , and R 3 each represents the same or different alkyl group, and R 1 , R 2 , and R 3 The total number of carbon atoms in the alkyl groups of R is 3 to 15. 4 is an alkylene group having 1 to 5 carbon atoms, and Q - is the group -COO - or the group -SO 3 - It is.]

2. In the general formula (1), R 1 , R 2 , and R 3 is an alkyl group having 4 or 5 carbon atoms, and R 4 The method according to claim 1, wherein is an alkylene group having 1 to 3 carbon atoms.

3. 3. The method according to claim 1, wherein the concentration of the compound represented by formula (1) in the aqueous solution is 0.3 to 10.0 M.

4. A chlorophyll-containing extract obtained by subjecting a plant part or algae containing chlorophyll to an extraction treatment using an aqueous solution containing a compound represented by general formula (1) as an extraction solvent. [Formula 1] [In the general formula (1), R 1 , R 2 , and R 3 each represents the same or different alkyl group, and R 1 , R 2 , and R 3 The total number of carbon atoms in the alkyl groups of R is 3 to 15. 4 is an alkylene group having 1 to 5 carbon atoms, and Q -is the group -COO - or the group -SO 3 - It is.]

5. An extraction solvent for extracting chlorophyll from plant parts or algae, comprising an aqueous solution containing a compound represented by general formula (1). [Formula 1] [In the general formula (1), R 1 , R 2 , and R 3 each represents the same or different alkyl group, and R 1 , R 2 , and R 3 The total number of carbon atoms in the alkyl groups of R is 3 to 15. 4 is an alkylene group having 1 to 5 carbon atoms, and Q - is the group -COO - or the group -SO 3 - It is.]

6. A method for producing solid chlorophyll, comprising the steps of: A step of diluting the chlorophyll-containing extract according to claim 1 with water to cause precipitation of chlorophyll; The method further comprises the step of recovering the precipitate.

7. 7. The method according to claim 6, wherein the concentration of the compound represented by general formula (1) contained in the extract is adjusted to 1.3 M or less by diluting the extract with water.

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