JPWO2018124216A1 - Method for producing lipid extract - Google Patents

Abstract

The present invention includes a step of obtaining a lipid-containing extract using one or more raw materials selected from the group consisting of land plants and algae using a hydrophilic organic solvent, and converting chlorophyll contained in the lipid extract into pheophytin. And a method for producing a lipid-containing extract substantially free of chlorophylls, comprising a step of removing pheophytin from the obtained lipid extract to obtain a lipid-containing extract. Provides a method for producing lipid extracts that are substantially free of chlorophyll without compromising the lipid components originally contained in plants and algae, and without using expensive adsorbents, large amounts of solvents, or expensive enzymes To do.

Description

The present invention relates to a method for producing a lipid extract. More particularly, the present invention relates to a method for producing a lipid extract that is substantially free of chlorophylls.
This application claims the priority of Japanese Patent Application No. 2016-254607 filed on Dec. 28, 2016, the entire description of which is hereby specifically incorporated by reference.

  Land plants and algae contain various lipid components. For example, fatty acids such as α-linolenic acid, hexadecatetraenoic acid, octadecatetraenoic acid, eicosapentaenoic acid, neoxanthine, violaxanthin, lutein Carotenoids such as fucoxanthin and β-carotene, and ceramides such as glucosylceramide are known as functional lipids.

  Studies on the functionality of plant-derived lipid components include the following. Fatty acids such as α-linolenic acid and eicosapentaenoic acid are allergic inhibitory effect (Non-patent document 1), pneumonia preventing effect (Non-patent document 2), blood lipid lowering action, blood pressure lowering action, antithrombotic action, anti-inflammatory. It has been reported to have an action and an anticancer action (Non-patent Document 3). Carotenoids such as neoxanthine have been reported to have an anti-obesity effect (Non-Patent Document 4), an anti-inflammatory action (Non-Patent Document 5), and a high apoptosis-inducing ability to cancer cells (Non-Patent Document 6). . Further, glucosylceramide has been reported to have a skin protecting effect, a skin moisturizing effect, an intestinal environment improving effect, and an antitumor effect (Non-Patent Documents 7 to 10).

  When a lipid component is extracted from a land plant or algae using a hydrophilic organic solvent, chlorophyll, a photosynthetic pigment, is extracted at the same time. For example, when a lipid component is extracted using spinach as a raw material with ethanol, the ethanol contains a lipid component and chlorophyll. Since chlorophyll promotes oxidation of lipids, if chlorophyll is not removed from the lipid extract, the quality of the lipid component-containing product may be deteriorated. Moreover, since chlorophyll decomposes into pheophorbide which is a causative substance of photosensitivity, it is not desirable to take a large amount. Therefore, when extracting a lipid component from a chlorophyll containing raw material and using for a foodstuff, a pharmaceutical, or cosmetics, it is necessary to remove a chlorophyll.

  There are the following methods for removing chlorophyll. Patent Document 1 discloses a method for removing chlorophyll from glyceride oil by treatment with an acid-treated amorphous silica adsorbent. In Patent Document 2, chlorophyll is added in two stages, decolorized by adding white clay to deoxidized oil obtained by deoxidizing crude oil by a conventional method, removing the green pigment in the deoxidized oil, and further decolorizing by adding activated carbon. A method of removing is disclosed. Patent Document 3 discloses a method of removing chlorophyll from a plant extract using hydrophobic chromatography. Patent Document 4 discloses a method of decolorizing enzymatically by allowing a chlorophyllase enzyme to act on a vegetable oil containing chlorophyll.

JP-A-63-310635 JP-A-56-21554 No. 2005/27937 (WO2005 / 027937) Japanese translation of PCT publication No. 2008-505619 (WO2006 / 009676)

Xie, N. et al., Archives of Medical Research, 2011; 42 (3): 171-81. Dewell, A. et al., The Journal of Nutrition, 2011; 141 (12): 2166-71. "AA, EPA, DHA-Highly unsaturated fatty acid" edited by Hikaru Kayama, Hoshiseisha Koseikaku Okada, T. et al., Journal of Oleo Science, 2008, 57, 345-351. Soontornchaiboon, W., Biological and Pharmaceutical Bulletin., 2012, 35, 1137-1144. Hosokawa, Bio Industry, 21 (2004) 52-57. Kazuhiko Ma, Oreoscience, Volume 7 (2007) No.4 p.141-149 Hamajima, H. et al., SpringerPlus, 2016, 5, 1321. Kawada, C. et al., Bioscience, Biotechnology, and Biochemistry, 2013, 77, 867-869. Kawano, K. et al., Phytotherapy Research, 2013, 27, 775-783. The entire descriptions of Patent Literatures 1 to 4 and Non-Patent Literatures 1 to 10 are specifically incorporated herein by reference.

  However, in the methods described in Patent Documents 1 and 2, not only chlorophyll but also carotenoids, which are lipid components, phospholipids and glycolipids are simultaneously removed from the lipid extract, and the yield of functional lipid components is reduced. It was causing a decline. In the method described in Patent Document 3, since an expensive adsorbent is used, it is not suitable for practical use in terms of economy, and a large amount of solvent is used, so there is a problem in safety. In the method described in Patent Document 4, since an expensive enzyme is used, there is a problem that it is not suitable for practical use in terms of economy.

  An object of the present invention is to provide a novel method for producing a lipid extract containing substantially no chlorophyll, which solves the problems of the conventional techniques. Specifically, lipid extraction that is substantially free of chlorophyll without excessively damaging the lipid components originally contained in plants and algae, and without using expensive adsorbents, large amounts of solvents, or expensive enzymes It is an object of the present invention to provide a method for manufacturing a product.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have converted chlorophyll contained in the extract into pheophytin and then removed pheophytin in a lipid extract obtained from an organic solvent of land plants and algae. The present inventors have found that it is possible to obtain a lipid extract containing substantially no chlorophylls, and have completed the present invention.

That is, the present invention is as follows.
[1] A step of obtaining a lipid extract from one or more raw materials selected from the group consisting of plants and algae using a hydrophilic organic solvent,
Lipid containing substantially free of chlorophylls, comprising converting chlorophyll contained in the lipid extract into pheophytin, and removing pheophytin from the lipid extract containing pheophytin to obtain a lipid-containing extract. A method for producing an extract.
[2] The hydrophilic organic solvent is selected from the group consisting of methanol, ethanol, butanol, propanol, diethyl ether, 1,3-butylene glycol, ethylene glycol, propylene glycol, dipropylene glycol, glycerol, acetone, and mixtures thereof. The production method according to [1].
[3] The production method according to [1] or [2], wherein the hydrophilic organic solvent is selected from the group consisting of ethanol and 1,3-butylene glycol, and a mixture thereof.
[4] The production method according to any one of [1] to [3], wherein the conversion of the chlorophyll to pheophytin is performed by acidifying the lipid extract.
[5] The production method according to [4], wherein the acidification of the lipid extract is performed by adjusting the lipid extract to pH 5.5 or lower.
[6] The production method according to any one of [1] to [5], wherein pheophytin is removed from the lipid extract containing pheophytin using diatomaceous earth.
[7] The production method according to [6], wherein the lipid extract containing pheophytin is contacted with diatomaceous earth in the presence of potassium chloride and / or sodium chloride.
[8] The production method according to [6], wherein the lipid extract containing pheophytin is contacted with diatomaceous earth in the presence of shell powder and / or calcium carbonate.
[9] The production method according to any one of [1] to [8], wherein the raw material is selected from kale, spinach, spirulina, and a mixture thereof.
[10] The production method according to any one of [1] to [9], wherein the lipid-containing extract contains 2.0% by weight or less of chlorophylls.

  According to the present invention, chlorophyll can be removed from a lipid extract without impairing lipid components originally contained in plants and algae. According to the present invention, a lipid extract containing no chlorophylls can be produced easily and efficiently.

Photomicrograph (400 times) observing adsorption of pheophytin by diatomaceous earth. After the ethanol extract of the kale juice juice residue was acidified and chlorophyll was converted to pheophytin, the ethanol concentration was adjusted to 40, 50, 60, 70 and 80 vol%. It can be seen that pheophytin is adsorbed very well on diatomaceous earth under the conditions of ethanol concentration of 50 to 70% by volume.

  The description of the present invention described below may be made based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In the present specification, a numerical range expressed using “to” means a range including numerical values described before and after “to” as a lower limit value and an upper limit value.

The present invention relates to a method for producing a lipid-containing extract substantially free of chlorophylls, comprising the following steps.
(1) A step of obtaining a lipid extract from one or more raw materials selected from the group consisting of plants and algae using a hydrophilic organic solvent,
(2) A step of converting chlorophyll contained in the lipid extract into pheophytin, and (3) a step of removing pheophytin from the lipid extract containing pheophytin to obtain a lipid-containing extract.

According to the method for producing a lipid-containing extract of the present invention, a lipid-containing extract substantially free of chlorophylls can be produced from a plant and / or algal raw material containing chlorophyll.
“Chlorophyll” is a pigment possessed by plants and algae that perform photosynthesis, and is also called chlorophyll. Typical chlorophylls include chlorophyll a, chlorophyll b, chlorophyll c1, chlorophyll c2, chlorophyll d, and chlorophyll f. In the present invention, chlorophyll is used as a general term.
In the present invention, “chlorophylls” are used to indicate both chlorophyll and pheophytin, which is a chlorophyll derivative. As will be described later, in the present invention, chlorophyll is converted to pheophytin for removal, and therefore pheophytin removal and chlorophyll removal are substantially synonymous in the present invention.

The “lipid-containing extract” and “lipid extract” of the present invention are extracts containing lipid components, and specifically, extracts containing lipid components derived from land plants and algae. Examples of the lipid component include fatty acids such as α-linolenic acid, hexadecatetraenoic acid, octadecatetraenoic acid, and eicosapentaenoic acid, and carotenoids such as neoxanthine, violaxanthin, lutein, fucoxanthin, and β-carotene. And ceramides such as glucosylceramide. The “lipid-containing extract” and “lipid extract” of the present invention are substantially free of chlorophylls.
In the case of substantially not containing chlorophylls, chlorophylls are not detected by the generally accepted detection method known to those skilled in the art, or even if it is detected, it is 2.0% by weight or less of the lipid-containing extract. In particular, it is more preferably 1.0% by weight or less. This is to prevent oxidation of fatty acids in the lipid-containing extract.

Process (1)
In step (1), a lipid extract is obtained from one or more raw materials selected from the group consisting of plants and algae using a hydrophilic organic solvent. After mixing the raw material and the hydrophilic organic solvent, this mixture may be filtered to remove solids, and a lipid extract may be obtained as a filtrate. The lipid extract contains chlorophyll together with lipid components.

  The plant that can be used as a raw material in the present invention is any organism selected from angiosperms, gymnosperms, ferns, and moss plants containing chlorophyll. When a plant is used as a raw material, the whole plant can be used, but it is particularly preferable to use leaves, petiole or stems. This is because leaves, petioles and stems often contain abundant functional lipid components together with chlorophyll. Algae that can be used as a raw material is not particularly limited, but is any organism selected from green algae, brown algae, diatoms, and yellow-green algae. Includes microorganisms having chlorophyll. When using algae as a raw material, all of the leaves, stems, and temporary roots can be used for large algae, and cells can be used as they are for microalgae. The raw materials are preferably plants and algae that are not toxic to the human body, or edible plants and algae. This is because lipid components extracted from these plants and algae are used for the purpose of maintaining and promoting health and beauty.

The raw material of the present invention is not limited, but in plants, for example, kale, spinach, cabbage, broccoli, cauliflower, beet leaf, barley young leaf, tomorrow leaf, mulberry leaf, soybean leaf, corn leaf, And algae such as Ginkgo biloba, for example, seaweeds such as Asakusanori, Proboscis, Kombu, Wakame, Akamoku, Hijiki, Aosa, etc., and microalgae such as Haematococcus, Spirulina, Chlorella, Euglena, Donariella Any one or a combination of two or more selected from the group consisting of these can be used. Examples of the above plants and algae are those that have been used as edible, health supplements or supplements. In addition to the above examples, plants and algae can be used as raw materials if they can extract lipid components that are particularly effective as functional components (health and beauty retention / maintenance components) by removing chlorophylls. can do.
Plants and algae used as raw materials in the present invention are preferably kale, spinach, barley young leaves, spirulina, chlorella, euglena, and donariella from the viewpoint of abundant eating experience and availability.
In the present specification, a plant may be described as a land plant, but this is a description considering that algae live in water. From plants that can be used as raw materials in the present invention, a place other than land (underwater It is not a description intended to exclude angiosperms, gymnosperms, ferns, and moss plants that grow on).

  Plants and algae that are raw materials can be used in any state such as raw, frozen, salted, and dried. Plants and algae that are raw materials can be used as they are, but can be used in any form such as pulverized product, shredded product, paste form, juice form, and the degree of pulverization can be adjusted and selected as appropriate. . For example, the plant and algae that are raw materials may be frozen with liquid nitrogen and ground in a mortar before mixing with the hydrophilic organic solvent. For example, the plant and algae that are raw materials may be mixed with a hydrophilic organic solvent, and then juiced with a mixer. The raw material is not particularly limited, and a squeezed residue generated during the production of green juice using kale or barley young leaves as a raw material can be used.

  The hydrophilic organic solvent used in the present invention is a water-soluble organic solvent or an organic solvent compatible with water. The hydrophilic organic solvent is not particularly limited and is selected from methanol, ethanol, butanol, propanol, diethyl ether, 1,3-butylene glycol, ethylene glycol, propylene glycol, dipropylene glycol, glycerol, acetone, and the like. One or a combination of two or more can be used. Ethanol, acetone, and 1,3-butylene glycol are preferable from the viewpoint of safety, and ethanol and 1,3-butylene glycol are more preferable from the viewpoint of ease of handling. The amount of the hydrophilic organic solvent used for lipid extraction can be appropriately determined in consideration of the type of the hydrophilic organic solvent and the types of plants and algae used as raw materials.

In the present invention, the lipid extract may contain water. When water is included, the concentration of the hydrophilic organic solvent in the lipid extract is determined based on the amount of water (other than solid content) originally contained in the plant and / or algae used as a raw material, water added by mess-up, etc. It means the volume% (v / v%) of the hydrophilic organic solvent when the total amount with the solvent is 100% by volume. In the present specification, the term “volume%” means v / v%.
In an embodiment of the present invention, when ethanol is used as the hydrophilic organic solvent, the ethanol concentration in extracting the lipid component in step (1) is not particularly limited. From the point of extraction of the lipid component, a certain amount, for example, 30% by volume or more of ethanol may be contained in the lipid extract. Although the present invention is not limited to a specific theory, the lipid component extraction efficiency may be affected by the lipid component content in the raw material and the pulverized state of the raw material rather than the ethanol concentration. The ethanol concentration at the time of lipid component extraction can be, for example, 50% by volume or more, and preferably 70% by volume or more. If the ethanol concentration at the time of lipid extraction in the step (1) is 40 to 70% by volume, there is an advantage that it is not necessary to adjust the ethanol concentration in the subsequent steps (2) and (3).

Process (2)
In step (2), chlorophyll contained in the lipid extract is converted to pheophytin.
In the present invention, the method for converting chlorophyll contained in the lipid extract into pheophytin is not particularly limited. For example, from the group consisting of the method of acidifying the lipid extract and the method of heating the lipid extract. You can choose. From the viewpoint of the stability of the lipid component, a method of adjusting the lipid extract to acidic is preferred. The method of acidifying the lipid extract is based on the extract obtained in step (1) from an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, an organic acid such as acetic acid, oxalic acid, malic acid, succinic acid or citric acid. It can implement by using the selected 1 type, or 2 or more types in combination. By acidifying the extract, Mg contained in the chlorophyll is released, and pheophytin in which Mg is substituted with 2 ═NH is generated. When converting chlorophyll to pheophytin, the pH of the lipid extract may be adjusted to 5.5 or lower, preferably in the range of pH 1-5, more preferably in the range of pH 2-4. This is because substitution of Mg contained in chlorophyll to = NH occurs easily under this pH condition.

Process (3)
In step (3), pheophytin is removed from the lipid extract containing pheophytin to obtain a lipid-containing extract.
In the production method of the present invention, any method for separating pheophytin from the lipid extract can be used to remove pheophytin from the lipid extract. The pheophytin may be solid-liquid separated as it is or may be separated by adsorbing the pheophytin to an adsorbent.
Examples of the adsorbent that can be used in the present invention include activated carbon, diatomaceous earth, activated clay, activated alumina, silica gel, and zeolite.
In a preferred embodiment of the present invention, diatomaceous earth is used for removal of pheophytin from the lipid extract, pheophytin is adsorbed on diatomaceous earth, and the diatomaceous earth adsorbed with pheophytin is removed from the extract by solid-liquid separation, whereby lipid-containing extraction is performed. You can get things. The diatomaceous earth that can be used in the present invention is not particularly limited, and diatomaceous earth sold by various companies such as a fired product, a flux fired product, and an acid-treated product can be used. The solid-liquid separation method is not particularly limited, and known methods such as vacuum filtration, pressure filtration, centrifugation, and filter press can be used.

  In the production method of the present invention, in order to favorably adsorb pheophytin contained in the lipid extract to diatomaceous earth, it is preferable to adjust the concentration of the hydrophilic organic solvent in the lipid extract. In addition to the hydrophilic organic solvent, the lipid extract mainly contains water derived from raw materials and water used for measuring up. When the hydrophilic organic solvent is ethanol, the ethanol concentration may be in the range of 60 to 80% by volume, or may be in the range of 65 to 75% by volume. From the viewpoint of chlorophyll removal efficiency, the ethanol concentration is preferably in the range of 40 to 70% by volume, and more preferably in the range of 50 to 70% by volume. When the organic solvent is ethanol, the present inventor has observed that pheophytin is very well adsorbed on diatomaceous earth, particularly under the condition of 50 to 70% by volume of ethanol (FIG. 1). The ethanol concentration may be adjusted before or after the conversion of chlorophyll contained in the lipid extract into pheophytin in step (2). The ethanol concentration is adjusted by adding distilled water when the ethanol concentration is high, and by adding high concentration ethanol when the ethanol concentration is low.

 In certain embodiments of the invention, a lipid extract containing pheophytin converted from chlorophyll can be contacted with diatomaceous earth in the presence of potassium chloride and / or sodium chloride. This is because the removal of pheophytin from the lipid extract is promoted by allowing diatomaceous earth to coexist with at least one compound selected from the group consisting of potassium chloride and sodium chloride in the lipid extract. Although the present invention is not limited to a specific theory, it is considered that pheophytin aggregation is promoted in the presence of inorganic substances such as potassium chloride and sodium chloride (salting out effect).

  In another embodiment of the present invention, a lipid extract containing pheophytin converted from chlorophyll can be contacted with diatomaceous earth in the presence of shellfish powder and / or calcium carbonate. This is because the removal of pheophytin from the lipid extract is promoted by allowing diatomaceous earth to coexist with at least one substance selected from the group consisting of shellfish powder and calcium carbonate in the lipid extract. The shell powder is not particularly limited, and for example, shell powder such as scallop and oyster can be used. Potassium chloride, sodium chloride and calcium carbonate are not particularly limited, and commercially available products sold by various companies can be used. The food additive grade is preferable from the viewpoint of safety.

  In step (3), a lipid-containing extract substantially free of chlorophylls is obtained. The lipid-containing extract obtained by the production method of the present invention may contain 2.0% by weight or less of chlorophylls, and particularly preferably 1.0% by weight or less of chlorophylls. The chlorophyll contained in the lipid-containing extract obtained by the production method of the present invention is particularly pheophytin, and its content is 2.0% by weight or less, particularly preferably 1.0% by weight or less.

Although the lipid extract in this invention is not specifically limited, It can be used for a foodstuff, a drink, feed, cosmetics, a pharmaceutical, etc.
Examples of the food include breads, confectionery, noodles, cooked rice, pasta, dressings, health foods, foods for the sick and elderly. The food for the above-mentioned sick or elderly includes semi-solid food and liquid food such as swallow food or food for persons with difficulty in chewing.
When a lipid extract is used for beverages, juices, milk beverages, alcoholic beverages, tea beverages and the like can be mentioned.
When the lipid extract is used for a feed, for example, a feed for pets, a feed for livestock or a feed for seafood can be mentioned.

When the lipid extract is used for cosmetics, it can be used as a moisturizing agent, a cosmetic agent and the like. These forms include emulsions, creams and emulsions.
When the lipid extract is used for pharmaceutical applications, for example, an anti-obesity agent, a blood sugar level increase inhibitor, a cancer cell growth inhibitor, an anti-inflammatory agent, etc. can be mentioned, and these forms include tablets, powders, capsules Various selections can be made as required.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Preservation of raw materials)
Kale Aojiru squeezed residue was frozen at -20 ° C after squeezing and stored at -20 ° C until use.
Spinach powder and dried spirulina were stored at room temperature in the dark until use.

(Measurement of water content)
Each raw material was dried at 105 ° C. for 24 hours, and the moisture content of the raw material before drying was calculated from the weight change before and after drying.
The moisture content of the kale juice extract, spinach powder, and dried spirulina used in Examples 1-11 and Comparative Examples 1-6 were 83.1%, 22.6%, and 4.3%, respectively. there were.

(Measurement of chlorophyll and pheophytin contained in lipid extract)
Measurements of chlorophyll and pheophytin were performed using HPLC. A calibration curve is made with each standard product of chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b (manufactured by Wako Pure Chemical Industries, Ltd.). Calculated.
Moreover, what added up chlorophyll content and pheophytin content was made into chlorophyll content.

Sample preparation for HPLC When using a kale juice extract as a raw material, the obtained lipid extract was dissolved in 30.5 ml of acetone to obtain a sample for HPLC. When spinach powder or dried spirulina was used as a raw material, the obtained lipid extract was dissolved in 40 ml of acetone to prepare a sample for HPLC. After sample preparation, the sample was immediately subjected to HPLC analysis.

HPLC conditions Pump: LC-20AD (Shimadzu Corporation)
Detector: Photodiode array spectrophotometer SPD-M20A (Shimadzu Corporation)
Column: Devesoil ODS-UG-5 (250 mmL × 4.6 mm ID) + guard column
ODS-UG-5 (10 mmL × 4.6 mm ID) (Nomura Chemical Co., Ltd.)
Column oven: CTO-20AC (Shimadzu Corporation)
Autosampler: SIL-20AC (Shimadzu Corporation)
Mobile phase: A (acetonitrile: methanol: water = 84: 9: 7, v / v / v)
B (methanol: ethyl acetate = 68: 32, v / v)
B concentration: 0% (8 min hold) -100% (concentration gradient 10% / min) -100% (10 min hold)
Sample injection volume: Kale Aojiru residue 40 μl
Spinach powder 5μl
2 μl of dried spirulina
Flow rate: 1.2 ml / min
Column temperature: 25 ° C
Detection wavelength: 431 nm (chlorophyll a), 465 nm (chlorophyll b)
666 nm (pheophytin a), 436 nm (pheophytin b)

(Example 1)
(Step of obtaining lipid extract)
Ethanol (special grade, manufactured by Wako Pure Chemical Industries, Ltd.) (600 ml) was added to 100 g of the thawed kale juice extract and left for 6 hours at room temperature in the dark to extract the lipid component. The ethanol concentration during the extraction was 88% by volume. During the standing, stirring was performed several times. After extraction, solid-liquid separation was performed by filtering with a filter paper (No. 2, manufactured by Toyo Filter Paper Co., Ltd.), and the filtrate was recovered. Next, the obtained filtrate was made up to 1200 ml with 88% aqueous ethanol solution to obtain a lipid extract.

(Process of converting chlorophyll to pheophytin)
Next, distilled water was added to 120 ml of the lipid extract to adjust the ethanol concentration to 60% by volume. The pH of the extract after adjusting the ethanol concentration was 6.2. 0.2 g of citric acid was added to a lipid extract having an ethanol concentration of 60% by volume. The pH of the extract after addition of citric acid was 3.4. After pH adjustment, the mixture was stirred for 1 hour at room temperature to convert chlorophyll to pheophytin. Chlorophyll was not detected by measurement using HPLC.

(Step of removing pheophytin to obtain a lipid-containing extract)
Next, 0.5 g of diatomaceous earth (# 100F, manufactured by Chuo Silica Co., Ltd.) was added to the acidified lipid extract, and the mixture was stirred for 1 hour at room temperature, followed by solid-liquid separation by vacuum filtration using filter paper. The obtained filtrate was distilled off at 50 ° C. using a rotary evaporator to obtain a lipid-containing extract.
(Analysis of pheophytin content)
The obtained lipid-containing extract, chloroform (50 ml), methanol (25 ml) and distilled water (15 ml) were dissolved in this order in a separatory funnel, and liquid-liquid distribution was performed. After liquid-liquid distribution, the lower layer (chloroform layer) was recovered. The obtained lower layer was distilled off at 50 ° C. using a rotary evaporator. Further, the solvent was completely distilled off with a nitrogen stream to obtain 0.0696 g of a lipid extract. The pheophytin content of the lipid extract was 1.5% by weight.

(Comparative Example 1)
The step of obtaining a lipid extract was performed in the same procedure as in Example 1.
Distilled water was added to 120 ml of the lipid extract to adjust the ethanol concentration to 60%. The mixture was stirred for 1 hour at room temperature, and the solvent was distilled off at 50 ° C. using a rotary evaporator to obtain a lipid-containing extract.
In order to analyze the chlorophyll content, the obtained lipid-containing extract, 50 ml of chloroform, 25 ml of methanol, and 15 ml of distilled water were sequentially dissolved in the separating funnel, and liquid-liquid distribution was performed. After liquid-liquid distribution, the lower layer was collected. The obtained lower layer was distilled off at 50 ° C. using a rotary evaporator. Further, the solvent was completely distilled off with a nitrogen stream to obtain 0.0734 g of a lipid extract. The chlorophyll content of the lipid extract was 9.2% by weight.

(Comparative Example 2)
Except not performing pH adjustment with citric acid, it carried out like Example 1 and obtained lipid extract 0.0640g. The chlorophyll content of the lipid extract was 6.5% by weight.

(Comparative Example 3)
Except not having added diatomaceous earth, it carried out like Example 1 and obtained lipid extract 0.0683g. The pheophytin content of the lipid extract was 4.8% by weight.

(Comparative Example 4)
A lipid extract 0.0652 g was obtained in the same manner as in Example 1 except that activated carbon (Taiko S type, manufactured by Phutamura Chemical Co., Ltd.) was used instead of diatomaceous earth. The pheophytin content of the lipid extract was 4.2% by weight.

(Example 2)
Chlorophyll was converted to pheophytin, and after adding 0.3 g of scallop shell powder (Hokkaido Industrial Technology Center), diatomaceous earth was added, and the same procedure as in Example 1 was performed to obtain 0.0582 g of a lipid extract. . The pheophytin content of the lipid extract was 0.8% by weight.

(Example 3)
Chlorophyll is converted to pheophytin, 0.3 g of calcium carbonate (special grade, manufactured by Wako Pure Chemical Industries, Ltd.) is added, and then diatomaceous earth is added. Obtained. The pheophytin content of the lipid extract was 0.5% by weight.

Example 4
Instead of citric acid, the same procedure as in Example 1 was performed except that 0.1 ml of 50% phosphoric acid (special grade, manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution was added, and 0.0572 g of lipid extract was added. Obtained. The pheophytin content of the lipid extract was 1.3% by weight. The pH of the extract after addition of phosphoric acid was 3.4.

  As shown in Table 1, the pheophytin content of the lipid extract in Example 1 was 1.5% by weight. On the other hand, the content of chlorophylls contained in Comparative Example 1 was 9.2% by weight, which was 6.1 times the content of pheophytin contained in Example 1. The content of chlorophylls contained in Comparative Example 2 was 6.5% by weight, which was 4.3 times the content of pheophytin contained in Example 1. The pheophytin content contained in Comparative Example 3 was 4.8% by weight, which was 3.2 times that of Example 1. From this, it was clarified that pheophytin can be removed by adjusting the pH to 5.5 or lower using citric acid, converting chlorophyll into pheophytin, and then contacting with diatomaceous earth. That is, it has been clarified that chlorophylls can be substantially removed. The pheophytin content contained in Comparative Example 4 was 4.2% by weight, which was 2.8 times that of Example 1. From this, it was revealed that using diatomaceous earth after converting chlorophyll to pheophytin is excellent in the effect of removing pheophytin. That is, it was revealed that diatomaceous earth is effective for removing chlorophylls.

From Example 2 and Example 3, it was revealed that the effect of removing pheophytin was further improved by adding scallop shell powder or calcium carbonate.
From Example 4, it was clarified that pheophytin can be removed by adjusting the pH to 5.5 or lower using phosphoric acid, converting chlorophyll into pheophytin, and then bringing it into contact with diatomaceous earth. That is, it has been clarified that chlorophylls can be substantially removed even when pH is adjusted using phosphoric acid.

(Example 5)
In the same procedure as in Example 1, a step of obtaining a lipid extract, a step of converting chlorophyll to pheophytin, and a step of removing pheophytin to obtain a lipid-containing extract were performed. In the step of converting chlorophyll to pheophytin, the ethanol concentration was adjusted to 60% by volume as in Example 1. The pH of the extract after addition of citric acid was 3.4, and chlorophyll was not detected by HPLC.
Finally, 0.0588 g of lipid extract was obtained. The pheophytin content of the lipid extract was 0.8% by weight.

(Example 6)
Extraction and separation were performed in the same manner as in Example 5 except that the step of converting chlorophyll to pheophytin and the step of removing pheophytin to obtain a lipid-containing extract were performed at an ethanol concentration of 70% by volume.
Finally, 0.0607 g of lipid extract was obtained. The pheophytin content of the lipid extract was 1.8% by weight.

(Example 7)
Extraction and separation were carried out in the same manner as in Example 5 except that the step of converting chlorophyll to pheophytin and the step of removing pheophytin to obtain a lipid-containing extract were performed at an ethanol concentration of 50% by volume.
Finally, 0.0507 g of lipid extract was obtained. The pheophytin content of the lipid extract was 1.0% by weight.

From the results of Table 2, it was revealed that the pheophytin concentration can be significantly reduced by carrying out the step of removing the pheophytin to obtain a lipid-containing extract at an ethanol concentration of 50 to 70% by volume of the extract. That is, it has been clarified that chlorophylls can be substantially removed.

(Example 8)
(Step of obtaining lipid extract)
To 100 g of dried Spirulina, 600 ml of ethanol was added, and the mixture was allowed to stand for 6 hours at room temperature in a dark place to extract lipid components. The ethanol concentration during extraction was 99% by volume. During the standing, stirring was performed several times. After extraction, solid-liquid separation was performed by filtering with filter paper, and the filtrate was recovered. Next, the obtained filtrate was made up to 1200 ml with 99 vol% ethanol aqueous solution to obtain a lipid extract.

(Process of converting chlorophyll to pheophytin)
Next, distilled water was added to 120 ml of the lipid extract to adjust the ethanol concentration to 60% by volume. The pH of the extract after adjusting the ethanol concentration was 6.3. 0.2 g of citric acid was added to the 60% ethanol concentration extract. The pH of the extract after addition of citric acid was 3.8. After pH adjustment, the mixture was stirred for 1 hour at room temperature to convert chlorophyll to pheophytin. Chlorophyll was not detected by measurement using HPLC.
(Step of removing pheophytin to obtain a lipid-containing extract)
Next, 0.5 g of diatomaceous earth was added, and the step of removing pheophytin by the same procedure as in Example 1 to obtain a lipid-containing extract was performed. 0.1495 g of lipid extract was obtained.
As a result of analysis, the pheophytin content of the lipid extract was 0.2% by weight.

(Comparative Example 5)
Extraction and separation were carried out in the same manner as in Example 8 except that the pH was not adjusted by adding citric acid to obtain 0.1543 g of a lipid extract. The chlorophyll content of the lipid extract was 6.2% by weight.

Example 9
Except that the step of converting chlorophyll to pheophytin and the step of removing pheophytin to obtain a lipid-containing extract were carried out in the same manner as in Example 8 except that the extraction and separation were performed in the same manner as in Example 8. 1785 g was obtained. The pheophytin content of the lipid extract was 0.5% by weight.

  As shown in Table 3, the pheophytin content of the lipid extract in Example 8 was 0.2% by weight. On the other hand, the content of chlorophylls contained in Comparative Example 5 was 6.2% by weight, 31 times that of Example 8. The pheophytin content in the case where the ethanol concentration was adjusted to 70% by volume (Example 9) was 0.5% by weight. From the results in Table 3, it was clarified that pheophytin can be removed by adjusting the pH to 5.5 or lower using citric acid, converting chlorophyll into pheophytin, and then contacting with diatomaceous earth. That is, it has been clarified that chlorophylls can be substantially removed.

(Example 10)
(Step of obtaining lipid extract)
To 100 g of spinach powder, 600 ml of ethanol was added, and the mixture was allowed to stand for 6 hours at room temperature in a dark place to extract lipid components. The ethanol concentration during the extraction was 96% by volume. During the standing, stirring was performed several times. After extraction, solid-liquid separation was performed by filtering with filter paper, and the filtrate was recovered. Next, the obtained filtrate was made up to 1200 ml with a 96% aqueous ethanol solution to obtain an extract.

(Process of converting chlorophyll to pheophytin)
Next, distilled water was added to 120 ml of the extract to adjust the ethanol concentration to 60% by volume. The pH of the extract after adjusting the ethanol concentration was 6.4. 0.2 g of citric acid was added to the 60% ethanol concentration extract. The pH of the extract after addition of citric acid was 3.4. After pH adjustment, the mixture was stirred for 1 hour at room temperature to convert chlorophyll to pheophytin. Chlorophyll was not detected by measurement using HPLC.
(Step of removing pheophytin to obtain a lipid-containing extract)
Next, 0.5 g of diatomaceous earth was added, and pheophytin was removed in the same manner as in Example 1 to obtain a lipid-containing extract. A lipid extract of 0.0928 g was obtained. As a result of analysis, the pheophytin content of the lipid extract was 0.6% by weight.

(Comparative Example 6)
Extraction and separation were carried out in the same manner as in Example 10 except that the pH was not adjusted by adding citric acid to obtain 0.0863 g of a lipid extract. The chlorophyll content of the lipid extract was 5.0% by weight.

(Example 11)
Chlorophyll was converted to pheophytin, and after adding 2 g of potassium chloride (special grade, manufactured by Wako Pure Chemical Industries, Ltd.), extraction and separation were performed in the same manner as in Example 10 except that diatomaceous earth was added. 0817 g was obtained. The pheophytin content of the lipid extract was 0.2% by weight. The pH after addition of potassium chloride was 3.4.

  As shown in Table 4, the pheophytin content of the lipid extract in Example 10 was 0.6% by weight. On the other hand, the content of chlorophylls contained in Comparative Example 6 was 5.0% by weight, which was 8.3 times that of Example 10. From this, it was clarified that pheophytin can be removed by adjusting the pH to 5.5 or lower using citric acid, converting chlorophyll into pheophytin, and then contacting with diatomaceous earth. That is, it has been clarified that chlorophylls can be substantially removed. From the results of Example 11, it was revealed that the effect of removing pheophytin was further improved by adding potassium chloride.

(Example 12: Analysis of fatty acid composition / content and carotenoid content in lipid extract)
A lipid extract was produced from the kale juice extract residue in the same manner as in Example 1 (lipid extract after chlorophyll removal treatment) and Comparative Example 1 (untreated lipid extract), and its fatty acid composition and content The amount as well as the content of carotenoids was analyzed by HPLC. The results are shown in Tables 5 and 6.

  From Tables 5 and 6, in the lipid extract that had been subjected to the chlorophyll removal treatment, the omega-3 highly unsaturated fatty acid α-linolenic acid (18: 3n) was compared with the lipid extract that had not been subjected to the chlorophyll removal treatment. -3) and carotenoids are not decreased. In the method for producing a lipid extract of the present invention, chlorophylls can be removed without impairing the content of the lipid component.

(Example 13: 1,3-butylene glycol is used instead of ethanol)
The kale green juice squeezed residue used in this example has a water content of 81.9% by weight, a lipid (α-linolenic acid) content of 34.7 mg / g dry weight, and a chlorophyll content of 3 .27 mg / g dry weight. These values were obtained by adding 10 times the amount of methanol to the kale juice juice residue and allowing it to stand overnight, followed by chloroform / methanol / water (10: 5: 3, v / v / v). It was obtained by analyzing the lipid extract obtained in addition.

After adding 8 g of 1,3-butylene glycol (hereinafter, 1,3-BG) (special grade, manufactured by Wako Pure Chemical Industries, Ltd.) to 5 g of Kale Aojiru squeezed residue, the mixture was stirred and then allowed to stand at 50 ° C. for 4 hours. Then, lipid components were extracted. A mixture of Kale Aojiru squeezed residue and 1,3-BG was filtered with filter paper (No. 2, manufactured by Toyo Roshi Kaisha, Ltd.), the solid matter was removed, and the filtrate was recovered to obtain lipid extract A. Two lipid extracts A were prepared, and one lipid extract A was used as a sample for measuring chlorophyll content and fatty acid content.
Next, 0.05 g of citric acid and 2 g of diatomaceous earth (# 100F, manufactured by Chuo Silica Co., Ltd.) were added to the remaining one lipid extract A, and the mixture was stirred at room temperature for 5 minutes. It was confirmed that the lipid extract after addition of citric acid was acidic (pH 5 or less) with a pH test paper. Solid-liquid separation was performed by filtration under reduced pressure using a filter paper to obtain lipid extract B.

For lipid extracts A and B, chlorophyll content and fatty acid content were measured by HPLC. In order to analyze the fatty acid content, margaric acid (17: 0) was used as an internal standard and a certain amount was added to the lipid extract. The main fatty acid in the kale residue lipid is α-linolenic acid (18: 3n-3) and hardly contains margaric acid (17: 0). The content of α-linolenic acid was determined from the ratio of α-linolenic acid and margaric acid having a fatty acid composition in the extraction solution.
Lipid extraction efficiency and chlorophyll removal rate in lipid extracts A and B based on lipid (α-linolenic acid) content and chlorophyll content of kale juice extract that has not undergone chlorophyll removal treatment Was calculated. The results are shown in Table 7.

As shown in Table 7, it can be seen that chlorophylls can be removed even if 1,3-butylene glycol is used instead of ethanol. The analysis result of lipid extract A showed that lipid can be extracted efficiently by using 1,3-BG. The analysis result of the lipid extract B showed that chlorophylls can be almost removed by carrying out the chlorophyll removal treatment. Further, by using 1,3-butylene glycol, the lipid extract can be used as it is as a cosmetic raw material without being concentrated.

  As described above, according to the present invention, when a lipid extract is produced using one or more selected from land plants or algae as a raw material, a hydrophilic organic material such as ethanol or 1,3-butylene glycol is used. The extract from which lipid components are extracted using a solvent is adjusted to acidity, chlorophyll is converted to pheophytin, and then contacted with diatomaceous earth to effectively remove pheophytin, which is easy and effective. In addition, it is possible to obtain a lipid extract having a pheophytin content of 2.0% by weight or less and substantially free of chlorophylls.

  The present invention relates to a method for producing a lipid extract. More specifically, the present invention relates to a method for producing a lipid extract from which chlorophyll is substantially removed, obtained by converting chlorophyll to pheophytin and then removing pheophytin. Although the lipid extract in this invention is not specifically limited, It can be used for a foodstuff, a drink, feed, cosmetics, a pharmaceutical, etc.

Claims (10)

A step of obtaining a lipid extract from one or more raw materials selected from the group consisting of plants and algae using a hydrophilic organic solvent;
Lipid containing substantially free of chlorophylls, comprising converting chlorophyll contained in the lipid extract into pheophytin, and removing pheophytin from the lipid extract containing pheophytin to obtain a lipid-containing extract. A method for producing an extract.   The hydrophilic organic solvent is selected from the group consisting of methanol, ethanol, butanol, propanol, diethyl ether, 1,3-butylene glycol, ethylene glycol, propylene glycol, dipropylene glycol, glycerol, and acetone and mixtures thereof; The manufacturing method according to claim 1.   The production method according to claim 1 or 2, wherein the hydrophilic organic solvent is selected from the group consisting of ethanol and 1,3-butylene glycol, and a mixture thereof.   The manufacturing method as described in any one of Claim 1 to 3 which converts the said chlorophyll into pheophytin by acidification of a lipid extract.   The production method according to claim 4, wherein the acidification of the lipid extract is performed by adjusting the lipid extract to pH 5.5 or less.   The production method according to any one of claims 1 to 5, wherein pheophytin is removed from the lipid extract containing the pheophytin using diatomaceous earth.   The production method according to claim 6, wherein the lipid extract containing pheophytin is contacted with diatomaceous earth in the presence of potassium chloride and / or sodium chloride.   The production method according to claim 6, wherein the lipid extract containing pheophytin is contacted with diatomaceous earth in the presence of shell powder and / or calcium carbonate.   The manufacturing method according to any one of claims 1 to 8, wherein the raw material is selected from kale, spinach, spirulina, and a mixture thereof.   The said lipid containing extract is a manufacturing method as described in any one of Claim 1 to 9 containing 2.0 weight% or less chlorophyll.