JPH08332080A - Enzymic treatment of dried chlorella - Google Patents

Abstract

PURPOSE: To provide a method for decomposing only the cell wall of a dried chlorella. CONSTITUTION: A dried chlorella is suspended in a buffer solution having a regulated osmotic pressure and treated with a cellulase and a pectinase as cell wall degrading enzymes to remove only the cell wall thereof. A polygalacturonase and a pectin-lyase are preferably used as the pectinase in combination. Furthermore, the dried chlorella, as necessary, is suspended in the buffer solution having the regulated osmotic pressure. The treatment for dispersing the dried chlorella may then be carried out and enzymic treatment may subsequently be performed. Ultrasonic treatment is preferred as the treatment for dispersing the dried chlorella.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for removing only the cell wall of dried chlorella containing a large amount of high quality proteins and fats and useful as a food material by a combination treatment of specific degrading enzymes to improve its digestibility. Regarding

[0002]

BACKGROUND ART Chlorella is a substantially spherical green single-celled alga having a diameter of 2 to 10 μm. Taxonomically, it is a green plant phylum, Chlorophyta, Chlorococcaceae, Oocystisidae,
Currently, about 20 species of chlorella are known. For mass cultivation, chlorella vulgaris (C. vulgaris),
Chlorella Regularis (C. regularis) is used. Outside, Chlorella ellipsoida (C.elli
psoidea), C. pyrenoidos
a), C. parasitica, C. conglomerata, C. variegata, C. autotrophica, and C. mue.
tabilis), chlorella nocturna (C. nocturna),
Chlorella photophila etc. are known. Since this chlorella has a high photosynthetic ability and an excellent component composition, it has been studied with the same green alga, Senedesmus, as a food source in the future, and is mass-produced in Japan.

The method of culturing chlorella includes autotrophic culture that is totally dependent on light and subtropical (heterotrophic) that utilizes organic carbon sources such as glucose and acetic acid.
hic) culture, mixed nutrition (m
There are three types of ixo-trophic) culture. Then, as the treatment after the large-scale culture, usually, after concentration by centrifugation, washing is repeated, and the enzyme in the chlorella cells is inactivated by heating and quenching at 100 ° C. for about 3 minutes to perform autolysis and denaturation of chlorophyll. In addition to preventing it, the digestibility at the time of eating is improved (blanching treatment), and finally a drying treatment is performed.

[0004] By the way, chlorella has low nutritional value, but its cell wall is very hard and digestion and absorption are poor, and its constituent components are largely different depending on species and strains, so that it has low value as food. It was evaluated. Then, the cell wall is hardly crushed by the ultrasonic treatment used for crushing general cells, and is decomposed by a normal cell-degrading enzyme,
Could not be dissolved. Alkaline (17.5% NaOH) containing almost no α-cellulose in recent years
A new species of Chlorella vulgaris has been developed (see Japanese Patent Publication No. 58-29074), which has a cell wall that is easily lysed with water and is easily mechanically disrupted. It has come to be effective not only in health foods but also in the treatment of atopic dermatitis and the like.

The methods of degrading and lysing only the cell wall that have been reported so far have been all about live chlorella. However, most raw chlorella (70
%) Is water, so that there are problems that it easily rots during transportation and storage and is difficult to handle. In order to solve this problem, dried chlorella, which has been dried and has a high degree of freedom in handling without fear of rotting, has come to be used, but in the case of dry chlorella, this is compared to the case of raw chlorella. Cell wall degradation is extremely difficult, and there were no cases where a method of degrading and lysing only the cell wall of dried Chlorella was examined.

[0006]

Under the above-mentioned technical background, the present invention has developed a method for decomposing and lysing only the cell wall while preventing leakage of the contents of the cells of dried chlorella, and developed a chlorella of a conventional species. By applying to Chlorella vulgaris E-25.

[0007]

DISCLOSURE OF THE INVENTION The inventors of the present invention have found that the treatment with cell wall degrading enzymes, cellulase and pectinase, is effective in removing the cell wall of dried chlorella. It came to completion. That is, the present invention relates to a method for treating dry chlorella, in which dry chlorella is suspended in an osmotic pressure adjusting buffer solution and treated with at least cellulase and pectinase as cell wall degrading enzymes to remove the cell wall. Further, the present invention is characterized in that dried chlorella is subjected to a treatment of suspending it in an osmotic pressure adjusting buffer solution to disperse the chlorella, and then treating it with cellulase and pectinase as cell wall degrading enzymes to remove only the cell wall. And a method for treating dry chlorella.

The cell wall degrading enzymes cellulase and pectinase that can be used in the present invention are known to be used for the preparation of plant protoplasts, and cellulase decomposes the cellulose microfibers that make up the majority of the primary cell wall to give pectinase. Breaks down the pectins that attach cells to each other. The greatest feature of the present invention is that cellulase or pectinase alone does not have a cell wall degrading effect in the dried chlorella of the present invention, and a combination of the two is indispensable. In addition, the purified enzyme has almost no effect of degrading the cell wall, and a crudely purified enzyme is preferable for decomposing a plurality of and multiple components in the cell wall.

As cellulase, the trade name cellulase
Onuluka RS (Cellulase OnozukaRS) (Yakult Honsha), Cellulase Onozuka R10 (Cellulase Onozuka)
a R10) (Yakult Head Office), Cellulase YC (Cellul
ase YC) (manufactured by Seishin Pharmaceutical), Meicelaze
(Meiji Seika), Driselase (Kyowa Hakko), and Cellulase TC (Cellulase TC)
va) and the like, and preferably cellulase Y obtained from Trichoderma viride.
C, cellulase Onozuka RS and R10. Particularly preferred is Cellulase Onozuka R10.

As pectinases, those containing polygalacturonase such as Macerozyme R10 (manufactured by Yakult Honsha) under the trade name, Pectolyase Y23 (manufactured by Seishin Pharmaceutical Co., Ltd.), Rohament P5 (Rohament P5) (Made by Serva), and those containing pectin lyase such as pectinase (made by Sigma) can be used, and preferred examples include macerozyme R10 and pectinase Y23.
Pectriase Y23 is an enzyme having a high activity, about 100 times higher than that of macerozyme R10, and it is a highly effective enzyme because it contains both polygalacturonase and pectin lyase. However, when the enzyme treatment time exceeds 2 hours, it may have a harmful effect on cells, and a combination system of pectriase Y23 and macerozyme R10 is particularly preferable.

Among the combinations of the cellulases and the pectinases, particularly preferred are the cellulases Onozuka R10, macerozyme R10, and pectolyase Y.
23 combinations. The cell wall-degrading enzyme according to the present invention is not limited to the above-mentioned trade name enzyme.

The preferred enzyme concentration of cellulase is 10 to 1
The amount is 00 mg / ml, particularly preferably 50 to 80 mg / ml. The preferred enzyme concentration of pectinase is 10 to 10.
120 mg / ml, particularly preferably 55-90 mg / ml
Is. For cellulases and pectinases, at a concentration below the lower limit, there is no effect of addition and no effect on the cell wall removal rate is observed, and when used at a concentration above the upper limit, the cell wall removal rate decreases conversely. From the viewpoint of the effect of cell wall removal rate, cellulase onozuka R10, macerozyme R10, and pectolyase Y23 were added to 60 mg / ml, 60 mg / ml, and 5, respectively.
When used at a concentration of mg / ml (the concentration ratio of cellulase and pectinase is 1: 1.1) is preferred.

The reaction between dry chlorella and the above-mentioned cell wall degrading enzyme is carried out by suspending dry chlorella in an osmotic pressure adjusting buffer solution so that individual cells are evenly dispersed, and using a cell wall degrading enzyme and shaking. Done. Preferred shaking conditions include 100 to 1 hours at a temperature of 20 to 37 ° C. for 1 to 48 hours from the viewpoint of effective action of the enzyme and uniform mixing of the solution.
A shaking speed of 50 rpm is preferred. With a shaking speed of 120 rpm, a shaking temperature of 30 ° C. and a shaking time of 24 hours are particularly preferable. In the present invention, the osmotic pressure adjusting buffer solution is a 25 mM potassium phosphate buffer solution having a pH of 6 to which an osmotic pressure adjusting agent such as sorbitol, sucrose or sodium chloride is added. The purpose of adding the osmotic pressure adjusting agent is to lower the external pressure (to make it hypotonic) as compared with the intracellular pressure and to prevent cells having lost the cell wall from easily rupturing and leaking the contents.

In the present invention, the dry chlorella used may be suspended in an osmotic pressure adjusting buffer solution and then treated in advance before the enzyme treatment to improve the dispersibility. This is because even if the dried chlorella is suspended in a buffer solution, the cells may stick to each other to form a large lump, and effective enzyme treatment cannot be performed. But,
If the cells themselves are disrupted by the pretreatment, nutrients will leak out unless the cells are freeze-dried and the properties of the food material cannot be maintained.Therefore, carefully consider the pretreatment conditions. is necessary.

Examples of the pretreatment method include a homogenizer treatment, a surfactant treatment, and an ultrasonic treatment. In the homogenizer treatment, dry chlorella is not dispersed in 1 minute or less, and destruction of chlorella is observed in 15 minutes or more under a microscope. Further, as the surfactant, Tween 20, Tween 40, Tween 60, Tween 80, and
Triton X-100 etc. can be used, a dispersed state can be obtained at a low concentration, and Tween 60 which is easy to remove by centrifugation
Is preferred. When the ultrasonic treatment time is 20 minutes or less, the dispersion of chlorella does not proceed. On the other hand, even if the ultrasonic treatment is performed for 30 minutes or more, the dispersion state is not improved, so that it is preferably about 30 minutes.

Considering the dispersibility of cells, the yield after treatment, and the hygiene in the future application to medicines and foods, ultrasonic treatment is the most effective pretreatment method. When it is desired to obtain only the dispersed cells, further filtration may be performed after this. Examples of the filtration method include a method using a glass filter and a method using a nylon mesh. When a glass filter is used in a large-capacity treatment, there is a problem that filtration cannot be performed due to clogging, and the use of nylon mesh is preferable.

The residual state of the cell wall of dried chlorella after the enzyme treatment was judged based on the observation result using a fluorescent reagent, calculation of osmotic pressure sensitive cells to sodium dodecyl sulfate solution (SDS solution), total sugar in the supernatant. It is performed by measuring the amount and observing with an electron microscope. Observation with a fluorescent reagent is performed by staining cells with a cell wall-binding fluorescent reagent that specifically binds to the cell wall and observing with a fluorescence microscope. Tested several fluorescent dyes, Fluorostein II
(Manufactured by Dojindo) was selected as the optimum reagent. When fluorescent dyes are bound to the cell wall (polysaccharide component), they are colored blue under fluorescent irradiation by fluorescein II staining. Therefore, chlorella cells with residual cell walls develop blue, while intracellular chloroplasts, etc. Tends to develop red color due to fluorescence, and red color is seen in chlorella cells whose cell wall has been removed by the action of an enzyme.

To calculate the osmotic pressure-sensitive cells, pure water or an SDS aqueous solution is added to the suspension of enzyme-treated cells, the number of cells remaining without rupture is measured, and the number of ruptured cells, that is, osmotic pressure sensitivity is measured. This is done by calculating the number of cells. This is because the SDS solution has a surface-active effect, and cells with depleted cell walls (or a large degree of depletion) are easily ruptured in an SDS solution containing no osmotic agent, and that of chlorella The plasma membrane is generally strong, and it utilizes the fact that even if the cell wall is lost, it hardly ruptures in the osmotic pressure adjusting buffer solution and maintains its original form.
Further, the apparent cell wall removal rate is calculated by the following formula. Cell wall removal rate = (n ₀ −n) / n ₀ n ₀ : Number of cells remaining in 25 mM potassium phosphate buffer solution n: Number of cells remaining in SDS solution The total sugar amount in the supernatant was measured by the enzyme. The treated cells are separated by centrifugation, and the total sugar content in the supernatant is measured by the phenol-sulfuric acid method or the like to examine the change.

[0019]

The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Determination of optimum enzyme concentration in dry chlorella treatment
A constant 0.5 g of dried chlorella [Chlorella pyrenoidosa (made by Taiwan Chlorella)] is used as an osmotic pressure regulator at 1.2M.
10 ml of osmotic pressure adjusting buffer solution dissolved in 25 mM potassium phosphate buffer solution (pH 6.0) containing sorbitol
Suspended in. After stirring with a magnetic stirrer for 15 minutes, the mixture was filtered with a nylon mesh having a pore size of 15 μm. After confirming that the obtained chlorella cells were uniformly dispersed in the suspension solution by observing with an optical microscope, the chlorella cells in the osmotic buffer solution had an initial concentration of 1.0 × 10 ⁸ /
Diluted to make up to ml. Cellulase in this solution
Onozuka R-10 (manufactured by Yakult Honsha), Macerozyme R
-10 (manufactured by Seikagaku Corporation) and Pectriaze Y-23
Enzymes (manufactured by Dojindo) were further mixed in various concentrations in combination. After reacting the enzyme for a predetermined time, 1 at 4 ° C
The precipitate was recovered by centrifugation at 0000 xg for 10 minutes. This is resuspended in osmotic buffer solution and kept at 4 ° C for 10
The enzyme solution was removed by centrifugation at 000 × g for 10 minutes, and a suspension of washed chlorella cells and cell debris was prepared and used to examine the removal efficiency of dry chlorella cell walls. The residual state of the chlorella cell wall was judged by the observation result using a fluorescent reagent and the result of the cell wall removal rate by adding the SDS solution.

By observation under a fluorescence microscope and comparison of cell wall removal rates, cellulase Onozuka R-10 at 60 mg / ml and macerozyme R under shaking conditions of 120 rpm.
-10 to 60 mg / ml and Pectriase Y-23 to 5 mg
The cell wall was most efficiently removed when the cells were mixed with each other / ml. At that time, the optimum temperature was 30 ° C. and the optimum treatment time was 24 hours. When dry Chlorella was treated under these optimal conditions, the number of cells was about 3 compared to before treatment.
Although it decreased by 0%, the cell wall removal rate reached 98%. In the observation under the fluorescence microscope, almost no cells having a cell wall remained. For comparison, the same experiment was carried out under the condition that the enzyme was not contained, but no cells with the cell wall removed were observed as compared with those before the treatment.

Example 2 Enzymatic Treatment of Dry Chlorella Under the optimal enzyme treatment conditions of Example 1, actual treatment of chlorella cells was performed. The amount of dry chlorella charged per enzyme solution was set to 40 mg / ml and used as it was without passing through a nylon mesh. The other conditions were the same as in Example 1. After the treatment, Chlorella was collected and observed under a fluorescence microscope, and it was found that the cell wall was removed from almost all the cells as in Example 1. Almost no cells remained when the SDS solution was added. The sample after enzyme treatment was freeze-dried, and this sample was suspended in pure water and 25 mM phosphate buffer solution and observed under a microscope. As a result, there were few aggregates compared to the original dry Chlorella, and it was dispersed in individual cells. There were many things. When the SDS solution was added to these resuspended preparations, almost no remaining cells were observed.

Example 3 By sonication of dry Chlorella
Cell disruption Dry chlorella is suspended in pure water or 25 mM potassium phosphate buffer solution (pH 6.0) to a concentration of 40 mg / ml, and stirred with a magnetic stirrer at room temperature for about 10 minutes to dry the water. Soaked in Chlorella. An ultrasonic wave generator (manufactured by Tomy Seiko Co., Ltd.) was oscillated at 30 kHz for 30 minutes while cooling to 4 ° C. with the tip of a vibrating part of an ultrasonic generator (Tomy Seiko Co., Ltd.). The ultrasonically treated dry chlorella was treated in the enzyme solution in the same manner as in Example 1 to examine the cell wall removal efficiency. Even when ultrasonic treatment is performed,
When observed under a fluorescence microscope, almost no cell wall remained was observed. In addition, when treated with the SDS solution, all of the cells after the enzyme treatment were ruptured or lysed, and none of them retained the cell morphology.

In the observation of the dried chlorella under the microscope after the ultrasonic treatment, about 60% of the cells were crushed by the ultrasonic treatment, and the dispersed state of the chlorella cells was improved. In addition, the proportion of cells remaining after feeding with the SDS solution was about 80% compared to that before sonication, and some of the chlorella cells were damaged in the cell wall or plasma membrane. I understand. Further, the sample that had been ultrasonically disrupted in pure water or a 25 mM potassium phosphate buffer solution was freeze-dried and the properties were observed. Compared to dry chlorella before treatment, the standard sample is closer to powder, and a suspended state can be easily obtained even when pure water is added. It was confirmed that it was improved. In addition,
Under the microscope, the Chlorella cells were the same as before freeze-drying.

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, cell wall can be effectively removed by treating dried chlorella with cellulase and pectinase which are cell wall degrading enzymes. It is a combination of cellulase and pectinase that has a great effect on cell wall removal of chlorella composed of a complex combination of multiple components,
As is clear from the examples, cellulase onozuka R1
0, macerozyme R10, and pectolyase Y23 exhibited the greatest effect when used in a crudely purified state, and most cell walls were removed. Moreover, the cells from which the cell wall has been removed retain their shape in the osmotic buffer solution. That is, in this enzyme treatment, the nutrients of the dried chlorella are present in the cells, and the properties as a food material are sufficiently maintained even after the enzyme treatment.

Since the chlorella of the present invention was a dried product,
When it is suspended in a solution, it is not always uniformly dispersed.
However, with the enzyme treatment, the enzyme acts on the cell wall of Chlorella and the partial decomposition proceeds, thereby reducing the Chlorella in which the cells are aggregated due to the physical adhesion state, and the dispersed state of Chlorella is good as a whole. Become. In addition, for effective removal of dry chlorella cell walls, it may be preferable to pretreat dry chlorella suspended in an osmotic pressure adjusting buffer solution to improve dispersibility and then to treat with enzyme. Compared with homogenizer treatment and treatment with a surfactant, the pretreatment method using ultrasonic treatment can obtain uniformly dispersed cells in an amount necessary for enzyme treatment in a relatively high yield and in a short time. . It was revealed that this ultrasonic treatment is effective not only for improving the dispersion of cells but also for damaging the cell wall of cells. Moreover, since this treatment is a physical treatment, it is a very convenient method when it is applied to medicines, foods, etc. in the future without the problem of chemical substance contamination such as chemical treatment.

From the above, it has been clarified that the enzyme treatment of the present invention makes it possible to obtain a dry chlorella in which the cell wall is removed and the digestibility is improved while maintaining the shape. Chlorella after removal of the cell wall is expected to have improved digestibility, and is extremely valuable when considering its application to medicines, foods and the like. Furthermore, the sonication alone can damage the cell wall of most cells, and in the future, if an enzyme such as a protease in addition to the enzyme used in the present invention is examined, further effect on digestibility can be expected. It may be used in various fields.

Claims (4)

[Claims] 1. A method for treating dry chlorella, which comprises suspending dry chlorella in an osmotic pressure adjusting buffer solution and treating it with at least cellulase and pectinase as cell wall degrading enzymes to remove only the cell wall. 2. The method for treating dry chlorella according to claim 1, wherein polygalacturonase and pectinlyase are used in combination as the pectinase. 3. Dry chlorella is suspended in an osmotic pressure adjusting buffer solution, subjected to a treatment for dispersing the dry chlorella, and then treated with cellulase and pectinase as cell wall degrading enzymes to remove only the cell wall. A characteristic method for treating dry chlorella. 4. The method for treating dry chlorella according to claim 3, wherein the treatment for dispersing the dry chlorella is ultrasonic treatment.