JP2009142267A - Method for pulverizing plants that have been unicellularized - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent reattachment between cells that occurs when a unicellular plant is concentrated, and to prevent damage to a cell membrane during lyophilization or resuspension of the obtained unicellular powder.
A first step of causing an intercellular substance-lytic enzyme to act on a plant, a second step of adding cyclodextrin to a suspension containing a unicellular plant obtained by the first step, A third step of concentrating the suspension containing cells dispersed in the second step by vacuum distillation concentration or centrifugation, and a suspension concentrated in the third step by freeze drying or spray drying A powdering method including a fourth step of powdering is used.
[Effect] Cyclodextrins gather around the cells of a plant that has been unicellularized to form micelles with hydrophilic groups on the surface, thus preventing reattachment between cells. In addition, since the cyclodextrin functions as a stabilizer even after pulverization, the state of the cell membrane is maintained.
[Selection] Figure 3

Description

  The present invention relates to a method for pulverizing a plant that has been unicellularized, and is capable of preventing cell aggregation and adherence that occurs during the concentration of a single cell suspension, and also obtained during lyophilization after concentration. The present invention also relates to a suitable powdering method capable of preventing the cell wall and the cell membrane from being damaged even when the single cell powder is resuspended.

  Traditionally, in the fields of so-called health foods and cosmetics, plants such as vegetables, fruits and medicinal herbs are made into single cells, thereby trapping functional components such as nutrients and pigments in the cells of the plant. The technology of making powder and commercializing it is used.

  Advantages of unicellularization include improved preservation of functional components by maintaining the state in which functional components are confined in cells, and reduction of alteration and loss such as oxidation of functional components that occur during processing. It is done. In particular, functional components that are easily altered during processing cannot be effectively used in a method of mechanically crushing and extracting using a mixer or the like, and can only be made into single cells without damaging the cell wall or cell membrane. Its functionality can be fully utilized.

  The production method for converting a plant into a single cell and pulverizing generally comprises the following two steps. The first step is a step of making the plant unicellular. Plants are composed of many cells adhered with intercellular substances, which are mainly polysaccharides consisting mainly of pectin and protopectin in which pectin is insolubilized, and enzymes that degrade this polysaccharide. Plants can be made unicellular by acting.

  When the intercellular substance is mainly composed of protopectin, pectinase is used as the intercellular substance lytic enzyme. Pectinase is a general term for enzymes that degrade pectin, and is a mixture of several types of enzymes such as polygalacturonase and pectin lyase. In addition, since the activity and distribution of each enzyme varies depending on the origin, such as Bacillus genus, Rhizopus genus, Aspergillus genus, and the characteristics change accordingly, various pectinases are commercially available.

  Next, a step of drying and pulverizing the unicellularized plant suspension is performed. For example, a plant unicellularized with pectinase becomes a suspension containing unicellularized cells. The plant cell wall has a basic skeleton made of cellulose embedded in a substrate made of a polysaccharide (matrix polysaccharide: hemicellulose, pectin, etc.). Pectin is an acidic polysaccharide, and homogalacturonan, rhamnogalacturonan I, rhamnogalacturonan II, apiogalacturonan, arabinan, galactan and the like are known.

  Therefore, in order to commercialize unicellularized plants as health foods and cosmetics, it is indispensable to perform a unicellularization treatment with pectinase and then dry and pulverize the suspension containing the unicellularized plants. . The pulverization of a unicellular plant is realized by spray drying or freeze drying.

Thus, several prior arts including the above steps have been proposed.
JP-A-9-75026

  For example, in the above-mentioned Patent Document 1, an intercellular substance-degrading enzyme mainly composed of protopectinase that dissociates strong protopectin with an enzyme isolated from a Rhizopus genus filamentous fungus is allowed to act on plants. A method for decomposing an intercellular substance into a single cell of a plant is disclosed.

In addition, Patent Document 2 below discloses a spice in which an intercellular substance-dissolving enzyme is allowed to act on a spice raw material to disintegrate the plant tissue by unicellularization or fine organization, and the intercellular substance is dissolved and removed. .
JP-A-7-23732

Conventionally, various methods have been proposed for pulverizing plant suspensions that have been unicellularized. For example, in the Patent Document 1, when the number of cells in single cell suspension 1ml decreases as ¹⁰⁵ to about ^6, after the correction of this solution to pH 7.0, to spray drying subjected spray dryer Thus, a method for obtaining a single cell powder is disclosed.

  In addition, in Patent Document 2, an enzyme such as protopectinase is allowed to act on a spice raw material to be single-celled and sieved, and then the resulting suspension is centrifuged to remove the supernatant, and the obtained paste-like form is obtained. A method is disclosed in which the precipitate is washed with water and then freeze-dried (freeze-dried) to dry it.

  However, as in Patent Document 1, when a suspension containing a unicellular plant is spray-dried and powdered, for example, heat of 120 ° C. or higher is applied, which adversely affects cell membranes and intracellular components. There was a problem that giving was inevitable.

  In addition, as in Patent Document 2, when using a method of centrifuging the suspension to remove the supernatant and concentrating and then pulverizing by freeze-drying, the cells are concentrated at the point of concentration to a paste-like precipitate. Since the distance between the single cells becomes short, there is a problem that the single cells aggregate and adhere to each other.

  In this way, if lyophilization is performed in a state where the single cells are aggregated and in close contact with each other, the cells are destroyed when pulverized and powdered after drying. Protection of functional components becomes impossible. Therefore, it was considered that the suspension was not concentrated or the concentration ratio was reduced, but the amount of the plant that was unicellularized obtained by one freeze-drying was reduced, and the cost increase was unavoidable and profitable. Since it cannot be removed, commercialization becomes difficult.

  In addition, the conventional production method has a problem that the cell membrane is damaged due to ice crystallization during freezing even when the cell wall is not reattached during concentration.

  Plant cells protect the cell interior with a cell wall composed of cellulose, hemicellulose, etc., and a cell membrane composed of a lipid bilayer. The cell wall protects the cell from physical external forces, and the cell membrane controls the entry and exit of substances inside and outside the cell. Therefore, in the unicellularized powder with the cell membrane damaged, even if the cell wall is not damaged, oxygen freely passes through the gap between the cell walls, so that the functional component in the cell gradually changes in quality. Similarly, volatile components are released out of the cell wall gap. In addition, the biggest problem due to cell membrane breakage is leakage of water-soluble components and volatile components to the outside of the cell, which is generated when unicellularized powder is suspended in water. These components quickly diffuse out of the cell when suspended, and all the merits of the single cell powder are lost.

  To summarize the above, the conventional techniques include the problem of cell aggregation and adhesion that occurs during concentration (hereinafter referred to as the first problem), and the re-suspension of lyophilized powder or the obtained unicellularized powder. There was a problem of damage to the cell membrane or the like (hereinafter referred to as the second problem).

  The present invention has been made to solve the above-described conventional first and second problems, and it is possible to prevent aggregation and adhesion between cells that occur when a unicellular plant is concentrated. And it aims at providing the optimal single cell-ized powder which can also prevent the damage of a cell membrane.

In order to achieve the above object, the method for pulverizing a plant subjected to unicellular treatment of the present invention comprises:
A first step of causing an intercellular substance-lytic enzyme to act on a plant;
A second step of adding cyclodextrin to the suspension containing the unicellularized plant obtained by the first step;
A third step of concentrating the suspension containing the cells dispersed in the second step by vacuum distillation concentration or centrifugation;
A fourth step of pulverizing the suspension concentrated in the third step by freeze drying or spray drying;
The most important feature point is to have

  The plant unicellularized powder of the present invention is a plant unicellularized powder obtained by the above-described powdering method, and the most important feature is that cyclodextrin is added as a cell membrane stabilizer.

  According to the present invention, when cyclodextrin is concentrated around a plant cell that has been unicellularized to form micelles and the surfaces become hydrophilic groups and electrically repel each other, when concentrated by vacuum distillation concentration or centrifugation. However, aggregation, adhesion, and reattachment of single cells are prevented, and it becomes possible to maintain the single cell state and powder without destroying the cell wall, thereby solving the first problem.

  In addition, since the cyclodextrin added in the second step of the powdering method of the present invention remains after concentration and functions as a cell membrane stabilizer, the single cell obtained at the time of lyophilization of the suspension or obtained Even when the powdered powder is resuspended, the good state of the cell membrane is maintained and damage can be prevented, so that the second problem can also be solved. Therefore, the unicellularized powder of the plant of the present invention can greatly reduce the deterioration and leakage of functional components inside the cells, particularly water-soluble components and volatile components, and further function until reaching the intestine, for example. By retaining the sex component in the cell, the function of the drug delivery system similar to that of the liposome preparation can be obtained.

  For example, various enzymes, which are useful functional proteins contained in plants, vaccine proteins expressed in transgenic plants, and the like are easily degraded by proteolytic enzymes contained in the stomach when ingested. When these work effectively in the intestines, they are already deactivated when they reach the intestines and cannot fully function as enzymes or vaccine proteins. However, in the present invention, cyclodextrin or a derivative thereof and, if necessary, one or more oily substances selected from polyhydric alcohol, α-tocopherol, cholesterol, carotenoid, edible oil, fatty acid, fatty acid ester, cholesterol ester Since the cell membrane is stabilized by adding, the enzymes and vaccines inside the cells are released from the stomach and released from the intestines. Therefore, the functionality of the enzyme or vaccine protein can be sufficiently exerted in the intestine.

  The object of the present invention is to solve the first and second problems by including a first step of causing an intercellular substance lytic enzyme to act on a plant, and a unicellular plant obtained by the first step. A second step of adding cyclodextrin to the suspension; a third step of concentrating the suspension containing cells dispersed in the second step by vacuum distillation concentration or centrifugation; and the third step. And a fourth step of pulverizing the suspension concentrated by the step of freeze-drying or spray-drying. Hereinafter, an example of the 1st-4th process of this invention is demonstrated concretely.

(First step)
The first step is a step of causing an intercellular substance lytic enzyme to act on the plant. As the intercellular substance lytic enzyme, for example, pectinase can be used. For example, when a plant is cut to a required size and then immersed in water, an enzyme solution prepared so that pectinase is at a predetermined weight% is added and subjected to an enzyme reaction, a suspension containing single cells is obtained. In addition, it is good also as a protoplast by decomposing | disassembling a cell wall simultaneously using a cell wall decomposing enzyme together. For example, cellulase can be used as the cell wall degrading enzyme. By the first step, the plant becomes a single cell suspension.

  Since this suspension contains residue, it is filtered through a 20 mesh (JIS) sieve to remove the residue. In the suspension thus obtained, single cell particles are precipitated over time and separated into two layers.

(Second step)
The second step is a step of adding cyclodextrin to the suspension containing the unicellularized plant obtained by the first step. Specifically, for example, a small amount of 0.1 to 5 parts by weight of cyclodextrin is added to 100 parts by weight of the suspension obtained in the first step and stirred well. Cyclodextrin is a kind of cyclic oligosaccharide in which 5 or more glucoses are bonded.

  Moreover, you may add a polyhydric alcohol with a cyclodextrin at a 2nd process. This is because the polyhydric alcohol also functions as a stabilizer for the cell membrane of the unicellular powder together with the cyclodextrin. Specifically, with respect to 100 parts by weight of a plant, 0.05 to 10 parts by weight of cyclodextrin and 0.005 to 5 parts by weight of a polyhydric alcohol are added and gently stirred over a sufficient time. In addition, since a residue is contained in suspension, it filters with a filter as needed.

  The polyhydric alcohol to be added can be selected from glycerin, ethylene glycol, propylene glycol, polyethylene glycol, diethylene glycol and the like. More preferably glycerin is used.

  The second step can be performed simultaneously with the first step. That is, the intercellular substance-dissolving enzyme and cyclodextrin may be added to the same reaction tank at the same time, and the order of addition to the reaction tank may be preceded by cyclodextrin.

  As the cyclodextrin to be added, any of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and derivatives thereof may be used. In the present invention, “cyclodextrin” includes derivatives thereof. Any of unbranched cyclodextrin, branched cyclodextrin (glucosyl cyclodextrin, maltosyl cyclodextrin, etc.) and cyclodextrin derivatives (dimethyl cyclodextrin, hydroxyethyl cyclodextrin, etc.) may be used.

  However, according to various studies by the present inventors, it has been found that γ-form cyclodextrin is more preferable because of its high solubility in water. It has also been found that maltosyl β cyclodextrin is more preferable for the first problem of preventing reattachment between cells.

  The inside of the unbranched cyclodextrin, the branched cyclodextrin, and the cyclodextrin derivative is hydrophobic (lipophilic) and the outside is hydrophilic. Since each cell particle forms a micelle having a hydrophilic group on the surface by cyclodextrin, each of the micellized cell particles is dispersed by electrical repulsion, and a suitable suspension in which each single cell is dispersed can be obtained.

  In the second step, one or more oily substances selected from α-tocopherol, cholesterol, carotenoids, edible fats and oils, fatty acids, fatty acid esters, and cholesterol esters may be used in combination with cyclodextrin and polyhydric alcohol. This is because the oil component also functions as a cell membrane stabilizer of the unicellular powder together with cyclodextrin and polyhydric alcohol. Here, the oily substance may be a cyclodextrin inclusion body, a physical mixture with cyclodextrin, or a state in which the inclusion body and the physical mixture are mixed. Specifically, 10 to 50 parts by weight of the oily substance is added to 100 parts by weight of cyclodextrin and gently stirred over a sufficient time. In the case of using as an inclusion body, cyclodextrin and the oily substance are first added to the inclusion body by a general method such as strong stirring, and then added to the unicellularized plant suspension. Stir gently over time. It was found that the stability of the cell membrane was maximized by using an α-tocopherol γ-cyclodextrin inclusion body adjusted so that the molar ratio was α-tocopherol: γ-cyclodextrin = 1: 2. ing. This is thought to be because the cyclodextrin inclusion body of the oily substance has higher binding properties with the cell membrane than the oily substance alone or the cyclodextrin simple substance.

(Third step)
The third step is a step of concentrating the suspension in which the single cells obtained in the second step are dispersed by centrifugation or vacuum distillation concentration. In the case of using centrifugation, for example, the suspension is centrifuged at 1500 G for 10 minutes to separate the micellar single cell particles, and then water is added to obtain a suspension in which the concentration rate is adjusted. When using vacuum distillation concentration, for example, the suspension is distilled and concentrated at 30 ° C. under reduced pressure using a vacuum distillation concentrator to obtain a concentrated suspension. In the present invention, the addition of cyclodextrin in the second step prevents cell-to-cell adhesion. Therefore, the conventional technique causes re-adhesion and causes cell wall destruction in the subsequent powdering step. The high concentration that has been achieved becomes possible.

(Fourth process)
The fourth step is a step of pulverizing the suspension concentrated in the third step by freeze drying (freeze drying) or spray drying (spray drying). When lyophilization is used, powdering is performed by, for example, pre-freezing the concentrated suspension at −30 ° C., and then drying in a vacuum freeze dryer for about 50 hours so that the final product temperature is 60 ° C. or lower. It is carried out by crushing by applying an appropriate pressure and hitting. By the above first to fourth steps, it is possible to obtain a powder for health foods and cosmetics while maintaining the unicellular state as it is.

  Hereinafter, tests 1 to 5 performed for confirming the effects of the present invention will be shown, and the present invention will be described more specifically. However, the present invention is not limited to the following examples.

[Test 1: Confirmation of anti-reattachment effect when single cell suspension is highly concentrated]
Compare the properties of powder obtained by freeze-drying by changing pectinase (MaceroTeam A (registered trademark): Yakult Yakuhin Kogyo Co., Ltd.) into single cells and changing the concentration conditions by vacuum concentration. did. In addition, in order to confirm the outflow amount of the content component from the cells in the obtained powder, carrots containing a large amount of pigment (β-carotene) were used.

(Enzyme treatment)
After cutting 100 g of carrots into 5 mm squares, immerse them in 300 g of distilled water and add an enzyme solution adjusted so that the ratio of the pectinase is 0.5% by weight based on the total weight of water and carrots. The suspension was reacted at 45 ° C. for 120 minutes with stirring (300 rpm). The residue was filtered through a 20 mesh (JIS) sieve to obtain a suspension containing single cells.

(concentrated)
About addition of the cyclodextrin to the suspension containing a single cell, it changed into conditions as follows and set it as the comparative example 1 and Examples 1-3.
Comparative Example 1: No cyclodextrin added Example 1: β-cyclodextrin (CAVAMAX (registered trademark) W7, Wacker-Chemie GmbH) 1 wt% added to the suspension Example 2: Suspension Addition of 1% by weight of γ-cyclodextrin (CAVAMAX (registered trademark) W8, Wacker-Chemie GmbH) to the liquid Example 3: Maltosyl β cyclodextrin (G2-β-CDTM, Yokohama International) to the suspension Bio-laboratory) 1% by weight added

  Each suspension was stirred well using a stirrer and allowed to stand for 1 hour. Under these four conditions, each was concentrated by distillation under reduced pressure to 2.5 to 5 times the weight of the suspension.

(freeze drying)
The concentrated suspension was allowed to stand for 24 hours in a refrigerator and then frozen in a freezer. This frozen suspension was dried by lyophilization to obtain a powder. 0.01 g of the obtained dry powder was taken, added again to 10 cc of water, stirred for 15 minutes using MICROTUBE MIXER MT360 (manufactured by TOMY), and the state of the cells was confirmed using a microscope. In addition, about the comparative example 1 and Examples 1-3, the test was done twice, respectively. The results are as shown in Table 1.

  When concentrating a unicellular plant suspension, as shown in FIG. 5 (a), when cells are concentrated without adding cyclodextrin as in Comparative Example 1, the cells are in close contact with each other (several cells). Was observed). In addition, since the cells are in close contact with each other, it may cause damage to the cell wall during freeze-drying or pulverization after drying. As shown in FIG. It was. This result indicates that if the content component is volatile, it is lost during grinding. FIG. 1 shows the entire photomicrograph of Comparative Example 1.

  On the other hand, as in Examples 1 to 3, in the case of the present invention in which the suspension to which a small amount of cyclodextrin was added was concentrated, the result that there was almost no adhesion between cells was obtained. Specifically, FIG. 2 is a micrograph of Example 1 to which β-cyclodextrin was added, FIG. 3 is a micrograph of Example 2 to which maltosyl β-cyclodextrin was added, and FIG. Fig. 3 shows a photomicrograph of Example 3 with the addition of dextrin. None of the cells are in close contact with each other, and the outflow of cell content components is reduced.

  In particular, in the case of maltosyl β cyclodextrin, the ratio of the weight (g) of the obtained dry powder to the weight (g) of the concentrated suspension is high even when the concentration is 10% or more. In addition to the lack of adhesion, the cells were uniformly dispersed, and favorable results were obtained.

  When cyclodextrin is added to the suspension, it is considered that the cyclodextrin includes carboxy groups of pectin and methyl esterified carboxyl groups present on the cell walls of the cells in the suspension. Since many hydroxy groups exist outside the cyclodextrin and show hydrophilicity, the cells repel each other in the liquid, and even when concentrated to a high concentration, the distance between the cells is maintained.

  The solubility of cyclodextrin in water is 14.5 g / 100 mL for α form, 1.8 g / 100 mL for β form, 23.2 g / 100 mL for γ form, and 162 g / 100 mL for maltosyl β cyclodextrin at 25 ° C. Β-form has the lowest solubility. In the above experiment, the γ form was better than the β form, and maltosyl β cyclodextrin was better than the γ form. The action obtained in the present invention is related to the solubility of cyclodextrin in water. It is thought that. In particular, it was found that the best results were obtained when maltosyl β cyclodextrin was used.

  However, since γ-cyclodextrin can be carried out at a lower cost than maltosyl β cyclodextrin, it is desirable to use γ-cyclodextrin when it is desired to obtain a good effect of a certain level or more while suppressing the cost as much as possible.

[Test 2-1: Measurement of cell membrane retention of unicellularized powder]
(Making unicellular powder)
Pectinase (Macero Team A (registered trademark): Yakult Yakuhin Kogyo Co., Ltd.): To 20 ml of enzyme reaction solution with 5% added, 10 g of Australian blueberry peel cut into 5 mm sides was added, and incubated at 37 ° C. for 5 hours to give a single cell A suspension was made and the residue was removed using a 20 mesh filter. Γ-cyclodextrin (CAVAMAX (registered trademark) W8, Wacker-Chemie GmbH), glycerin (Wako Pure Chemical Industries, Ltd.) per 1 ml of this suspension (the number of red cells containing anthocyanin: 3.5 × 10 ³ / ml) Special grade), α-tocopherol (dl-α-tocopherol, manufactured by Wako Pure Chemical Industries, Ltd.) is added in an amount changed as in Examples A to D below so that it becomes uniform over time. After gently stirring, the supernatant was removed by centrifugation (1500 G × 10 minutes), and the remaining precipitate was lyophilized to obtain a unicellularized powder. In addition, cyclodextrin, glycerin, and α-tocopherol were not added at all, and a unicellular powder was prepared in exactly the same manner as Comparative Example a. Further, cyclodextrin, glycerin and α-tocopherol were not added at all, dextrin (Amicol R: manufactured by Nissho Chemical Co., Ltd.) was added, and other unicellular powders were prepared in exactly the same manner as Comparative Example b.

(Amount added to 1 ml of suspension)
Example A γ-cyclodextrin 1.0%
Example B γ-cyclodextrin 1.0%
Glycerin 0.1%
Example C γ-cyclodextrin 1.0%
Glycerin 0.1%
α-Tocopherol 0.2%
Example D α-Tocopherol γ-Cyclodextrin
Inclusion body (α-tocopherol: cyclodex
Thrin weight ratio = 1: 5) 1.0%
Glycerin 0.1%
Comparative Example a No addition Comparative Example b Dextrin 1.0%

(Cell membrane retention)
Each powder obtained as described above was suspended in 1 ml of distilled water, and among the anthocyanin-containing red cells, the number of cells whose cell membrane was not damaged was counted under a microscope. As an example of the anthocyanin-containing red cells in which the cell membrane is not damaged, the cells when the powder of Example D is resuspended in water are shown in FIG. The results are shown in Table 2 below.

  As is clear from Table 2, in the case of Comparative Example a powdered without adding anything and Comparative Example b powdered with dextrin, 10% of the cells retaining the cell membrane were resuspended when the powder was resuspended. In Example A, the amount of red cells retaining the cell membrane was significantly increased to 50% or more of the whole, and the cell membrane was stabilized in Example A by adding a small amount of γ-cyclodextrin. It was confirmed. Furthermore, in Examples B, C and D, by adding a small amount of glycerin and α-tocopherol together with cyclodextrin, the red cells holding the cell membrane become 70% to 90% of the total, and the stability of the cell membrane is further increased. Confirmed to do. Furthermore, by comparing Examples C and D, it was confirmed that the inclusion stability of α-tocopherol γ cyclodextrin was higher than that of a physical mixture of α-tocopherol and γ cyclodextrin.

[Test 2-2: Measurement of cell membrane retention]
(Making unicellular powder)
Scandinavian fruit and vegetable blueberry (10 g), intercellular substance lytic enzyme (Pectriase Y-23, manufactured by Shengshin Kogyo Co., Ltd .: 0.5%), γ-cyclodextrin (CAVAMAX (registered trademark) W8, Wacker-Chemie GmbH): 0.5% glycerin (special grade, manufactured by Wako Pure Chemical Industries, Ltd.): It was immersed in 30 ml of a solution containing 0.1%, and incubated at 40 ° C. for 1 hour to obtain single cells. After removing the residue from the reaction solution with a 20 mesh (JIS) filter, the residue was concentrated by centrifugation (1500 G × 10 minutes) and then freeze-dried to obtain 0.2 g of blueberry unicellularized powder according to the present invention. (Hereinafter referred to as Example 4). As a comparative control, a unicellularized powder obtained by the same method as above except that γ-cyclodextrin and glycerin were not added was used (hereinafter referred to as Comparative Example 2).

(Investigation of intracellular anthocyanin retention rate)
If the cell membrane is damaged, water-soluble components such as anthocyanins easily flow out when the unicellularized powder is suspended in distilled water. Therefore, in Example 4 and Comparative Example 2, the intracellular anthocyanin retention rate = ((total anthocyanin amount) − (leaked anthocyanin amount)) / (total anthocyanin amount of unicellular powder) was investigated.

  The amount of leaked anthocyanins was calculated from the amount of anthocyanins in the supernatant separated by centrifugation (1500 G) after suspending 0.05 g of unicellularized powder in 1 ml of distilled water and allowing to stand at 4 ° C. overnight. . The amount of anthocyanin was calculated from the absorbance at the maximum absorption wavelength of delphinidin extracted by heating at 100 ° C. for 1 hour with 1% hydrochloric acid acidic methanol. The test results are as shown in Table 3.

  As is clear from Table 3, the single-cellized powder of Example 4 according to the present invention has a cell membrane that is stabilized by the addition of γ-cyclodextrin and glycerin. It was confirmed that the leakage of the sex component to the outside of the cell was much reduced and the anthocyanin content as a powder was also improved by 10%.

[Test 3: Investigation of the stability of vitamin C in unicellularized powder]
(Making unicellular powder)
Pectinase (Maceroteam A (registered trademark): Yakult Yakuhin Kogyo Co., Ltd.) was allowed to act on 10 g of fruit and vegetable paprika, and 0.13 g of paprika unicellular powder was obtained under the same conditions as in Test 2-2 (hereinafter referred to as “paprika single cell powder”). Example 5). For comparison, paprika powder (hereinafter referred to as Comparative Example 3) obtained by pulverizing and freeze-drying paprika was used. As a comparative control, data of L-ascorbic acid (manufactured by Nacalai Tesque: purity 99.5%, hereinafter referred to as Comparative Example 4) was also measured.

(Acceleration test and vitamin C determination)
Paprika is rich in vitamin C (ascorbic acid). Vitamin C is protected and stabilized by the cell membrane as the cell membrane remains in good condition without being damaged. Therefore, the powders of Example 5, Comparative Example 3 and Comparative Example 4 were subjected to an accelerated test for 3 days under the conditions of 5% humidity and 45 ° C. The amount of vitamin C remaining per 0.01 g of each powder (%: amount of residual vitamin C after accelerated test / vitamin C amount before accelerated test) was determined. The results were as shown in Table 4 below.

  As is clear from Table 4, since the unicellularized powder according to the present invention maintains a good state without damaging the cell membrane, the intracellular vitamin C is lyophilized in Comparative Example 3 or Comparative Example 4. Compared with L-ascorbic acid, it was confirmed that it was much more stable.

[Test 4: β-carotene dissolution test]
Gastric juice treatment or intestinal juice treatment was performed using 0.05 g of the paprika single cell powder of Example 5 prepared in Test 3, and a β-carotene elution test was conducted. As a comparative control, 0.5 g of paprika fruits and vegetables (water content 90%), which were extremely chopped, was used as a specimen (hereinafter referred to as Comparative Example 5).

  The gastric juice used was 50 ml of the first solution of the Japanese Pharmacopoeia containing 4% pepsin, and the intestinal fluid was 50 ml of the second solution containing 2% trypsin and 2% pancreatin. The gastric juice treatment was incubated at 37 ° C. for 2 hours, and the intestinal juice treatment was incubated at 37 ° C. for 3 hours.

  Thereafter, the absorbance at the maximum absorption wavelength (453 nm) of β-carotene was measured, and the absorbance values of the eluted β-carotene were compared. The results were as shown in Table 5 below.

  In the paprika of Comparative Example 5, the gastric juice treatment was 0.1225 with respect to the total elution amount of 0.1736, and 70% of the total amount was eluted by the gastric juice treatment. On the other hand, in the paprika single cell-ized powder according to the present invention, the total elution amount is 0.7570, the intestinal fluid treatment is 0.5807, and 76% is eluted by the intestinal fluid treatment. From this result, it was clarified that when the paprika single-cellized powder of the present invention is orally ingested, β-carotene in the cell has a function of a drug delivery system mainly eluting in the intestine. Furthermore, since the paprika single cell powder of the present invention eluted β-carotene four times as much as the fruits and vegetables, it was confirmed that the digestibility was remarkably increased as compared with the fruits and vegetables.

[Test 5: Protein digestion test]
Using a ginseng unicellularized powder obtained in exactly the same manner as in Test 2-2 (hereinafter referred to as Example 6), a protein digestion test in the stomach and intestine was performed.

  Gastric juice and intestinal fluid were prepared in the same manner as in Test 4. The ginseng unicellular powder of Example 6 is suspended in gastric juice or intestinal fluid, incubated at 37 ° C. for 1 hour, and the amount of protein remaining in the gastric juice and intestinal fluid is determined, and leucine is quantified as an amino acid released in the treatment fluid. Then, the degradation rate with respect to the protein before digestion and the change over time in the amount of leucine were investigated. The result was as shown in the graph of FIG.

  As shown in the graph of FIG. 6, the ginseng unicellularized powder of Example 6 did not progress in protein degradation during gastric juice treatment, and the amount of amino acid (leucine) released by protein degradation almost changed between 15 and 60 minutes. do not do. On the other hand, in the intestinal juice treatment, protein degradation proceeds, and the amount of free amino acid (leucine) increases corresponding to the degradation. The proteolysis rate in the gastric juice treatment for 1 hour was less than 10% of the total amount, and the proteolysis rate in the intestinal fluid treatment was 33% of the total amount. From this result, when the unicellularized powder of the present invention is orally ingested, the stability of intracellular protein is maintained in the stomach, the cell membrane is disturbed in the intestine, and the function of the drug delivery system in which the protein is released and degraded. It was confirmed to have

  As described above, according to the present invention, the cyclodextrin is collected around the cells of the unicellular plant to form micelles, the surfaces become hydrophilic groups and electrically repel each other. Even when concentrating by separation, aggregation, adhesion, and reattachment of single cells are prevented, and it becomes possible to maintain a single cell state and powder without destroying the cell wall, thereby solving the first problem. it can.

  In addition, since the added cyclodextrin functions as a cell membrane stabilizer in the unicellularized powder of the present invention, the cell membrane can be used even when the suspension is lyophilized or when the obtained unicellularized powder is resuspended. Damage can be prevented and the second problem can be solved. In addition, according to the present invention, the following effects (1), (2), and (3) can also be obtained.

(1) Availability of spray drying The present invention can be powdered while maintaining the intracellular state even when spray drying is used as well as freeze drying. This is because, in the case of spray drying, for example, heat of 120 ° C. or higher is applied, and thus there has been a problem that the conventional powdering method inevitably adversely affects the cell membrane and intracellular components. This is because in the invention, cyclodextrin aggregates around a unicellular cell to form a membrane, so that the heat transfer into the cell is blocked as much as possible by this cyclodextrin membrane.

(2) It is possible to avoid cell-cell adhesion that occurs after becoming a powdered product. Since water-soluble polysaccharides present on the cell wall surface of unicellular plants are hygroscopic, conventional powdering methods use powders. Even after the product was made, there was a problem that the cell wall surface contained water and the cells adhered to each other over time. However, in the present invention, since the cyclodextrin itself is hygroscopic, the cyclodextrin absorbs water in place of the water-soluble polysaccharide present on the cell wall surface, so that the cell wall surface contains water as much as possible. The Therefore, in the present invention, the problem that cells adhere to each other after becoming a powdered product is solved, and the product can be handled easily.

(3) What can be used as a drug delivery system? A cell membrane is not decomposed in the stomach environment. Since the unicellularized powder obtained by the present invention is stabilized without damaging the cell membrane, it reduces deterioration and loss of natural functional components of the plant and prevents decomposition in the stomach when taken orally. Only after reaching the small intestine can the cell membrane be disturbed to release intracellular components. Therefore, especially about the functional component which loses an effect remarkably by alteration by gastric acid, the absorption rate in an intestine will improve and the effective utilization rate will be improved markedly.

  The present invention is not limited to the above embodiments, and the embodiments may be changed as appropriate within the scope of the technical idea described in each claim.

  The powdering method of the present invention is applicable to health foods and cosmetics made from various plants as raw materials, for example, regardless of plant types such as chili, bitter gourd, aloe, garlic, green papaya, soybeans, onions, turmeric, and longevity grass. Widely applicable.

The dry powder obtained by the pulverization method of Comparative Example 1 without adding cyclodextrin was photographed with an optical microscope (100 times). The dried powder obtained by the powdering method of Example 1 in which 1% by weight of β-cyclodextrin is added to the suspension is photographed with an optical microscope (100 ×). The dried powder obtained by the powdering method of Example 2 in which 1% by weight of γ-cyclodextrin is added to the suspension is photographed with an optical microscope (100 times). The dried powder obtained by the pulverization method of Example 3 in which 1% by weight of maltosyl β cyclodextrin is added to the suspension is photographed with an optical microscope (100 ×). The dry powder obtained by the pulverization method of Comparative Example 1 without adding cyclodextrin was photographed with an optical microscope (100 times), (a) shows a state in which cells are in close contact, and (b) shows content components. Shows a state in which the spilled into a lump. It is the graph which showed the result of the digestion test (test 5) of a protein, and showed the change for every time of the amount of protein remaining, and the amount of free leucine. It shows the anthocyanin-containing red cells in which the cell membrane was not damaged when the powder of Example D was resuspended in water in the measurement test of cell membrane retention (Test 2-1).

Claims (9)

A first step of causing an intercellular substance-lytic enzyme to act on a plant;
A second step of adding cyclodextrin to the suspension containing the unicellularized plant obtained by the first step;
A third step of concentrating the suspension containing the cells dispersed in the second step by vacuum distillation concentration or centrifugation;
A fourth step of pulverizing the suspension concentrated in the third step by freeze drying or spray drying;
A method for pulverizing a plant that has been unicellularized, comprising:   The method for pulverizing a plant subjected to unicellular treatment according to claim 1, wherein the cyclodextrin is γ-cyclodextrin.   The method for pulverizing a unicellularized plant according to claim 1, wherein the cyclodextrin is maltosyl β cyclodextrin.   A plant unicellularized powder obtained by the powdering method according to claim 1, wherein cyclodextrin is added as a cell membrane stabilizer.   The plant unicellularized powder according to claim 4, wherein the cyclodextrin is γ-cyclodextrin.   A plant unicellularized powder obtained by the pulverization method according to claim 1, wherein cyclodextrin and polyhydric alcohol are added as stabilizers for the cell membrane.   The plant unicellularized powder according to claim 6, wherein the polyhydric alcohol is glycerin.   A plant unicellular powder obtained by the powdering method according to claim 1, wherein the cell membrane stabilizer is selected from cyclodextrin, α-tocopherol, cholesterol, carotenoid, edible oil, fatty acid, fatty acid ester, cholesterol ester A plant unicellular powder characterized by adding one or more oily substances.   The plant unicellularized powder according to claim 8, wherein the oily substance is α-tocopherol.