JPH1076155A - Non-lipid soluble substance-containing microcapsule and method for producing the same - Google Patents

Abstract

(57) [Problem] To provide a yeast microcapsule having a high content of a non-lipid soluble substance. SOLUTION: A method for producing a microcapsule characterized by kneading a muddy yeast cell and a non-lipid-soluble substance, and a method in which an externally added non-lipid-soluble substance is contained in the yeast cell at 10% by weight or more. Microcapsules.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a microcapsule using a non-fat-soluble substance externally added as a core substance and a cell wall of yeast as an encapsulating substrate, and a method for producing the same.

[0002]

2. Description of the Related Art In a narrow sense, a microcapsule is a fine particle having a size of 1 μm to several hundred μm, which contains a useful substance in various states and characteristics and is uniformly covered with a thin film. As a production method thereof, a coacervation method, an interfacial polymerization method, an in situ method and the like are known as effective methods. In a broad sense, as represented by microspheres and the like, they are in the form of fine particles, but those in which a useful substance and an encapsulating base material are merely mixed and not necessarily completely covered are also used. Included in the concept of capsule. At present, microcapsules containing colorless and colored dyes, pharmaceuticals, agricultural chemicals, flavors, feed materials, food materials, and the like have been industrially manufactured.

[0003] On the other hand, there have been proposed several microcapsules utilizing microorganisms which are completely different from the above-mentioned production methods. Microbial microcapsules are obtained by encapsulating a core substance in an already-formed encapsulating substrate, in order to utilize the cell wall of the microorganism as an encapsulating substrate. Microbial microcapsules have the advantage over microcapsules made by other processes that they have a uniform particle size and that the encapsulated substrate is natural and safe. However, the substances to be included are generally limited to fat-soluble (US Pat. No. 400001480;
No. 107189, JP-B-7-032870).

At present, as a typical means for microencapsulating a non-lipid-soluble substance, there is a method in which a core substance is directly coated with a wax or the like. A substrate is required,
Disadvantages such as extremely poor dispersibility in water and poor palatability for use in food feed have been pointed out.

[0005] Although a product of a concept different from that of microbial microcapsules, a non-lipid soluble substance is contained in yeast cells at a high content by utilizing the metabolic activity of yeast, which is disclosed in US Pat. No. 3,986,933. 61-274676
No., JP-A-03-91477, JP-A-03-1834
No. 74. All of these substances and production methods are characterized by the fact that yeast cells themselves produce non-lipid-soluble substances, such as improved culture methods and breeding by selecting mutant strains. However, the types of non-lipid soluble substances that can be contained in these yeast cells at a high content rate are limited, and their application range is narrow.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a microcapsule which overcomes the above-mentioned disadvantages of the prior art and a method for producing the same.

[0007]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, have obtained muddy yeast cells in which extracellular water has been removed from the yeast cell slurry. ,
By kneading the muddy yeast cells and the non-lipid-soluble substance, a microcapsule having a high content of the non-lipid-soluble substance was successfully produced, and the present invention was completed. That is, the present invention provides a method for producing microcapsules, which comprises kneading muddy yeast cells and a non-lipid-soluble substance. The present invention also provides a yeast microcapsule containing a non-lipid-soluble substance added externally in an amount of 10% by weight or more inside yeast cells.

Hereinafter, the present invention will be described in detail. In the present invention, yeast cells are used as the encapsulation base material.
Yeast is a general term for microorganisms that grow by budding or division, and any yeast may be used in the present invention. Brewery yeast, baker's yeast, Torula yeast, and the like can be used, and specifically, for example, Saccharomyces cerevische of the genus Saccharomyces (Saccharomyces)
cerevisiae), Saccharomyces luxi (Saccharo)
myces rouxii, Saccharomyces carlsbergensis, Candida utilis, Candida tropicalis, Candida lipolytica, and Candida flaveri ) Etc. can be used. These can be used alone or in combination. There are various shapes of yeast depending on the type, but preferably a shape as close to spherical as possible. Further, the particle size is preferably in the range of 1 to 20 μm. These yeasts used in the present invention may be live yeasts, but those obtained by solubilizing and removing intracellular components of the live yeasts by enzyme treatment or the like (for example, autolyzed yeasts) are preferable. (Acid-treated yeast) is preferred. The yeast cells used in the present invention have a muddy appearance, and such muddy yeast cells are obtained from a yeast cell slurry (for example, a tank sediment after completion of beer fermentation). And its solids content is preferably about 20 to 30%, particularly preferably about 22 to 25%. Examples of the method for removing extracellular water from the yeast cell slurry include centrifugation at 4500 rpm for 10 minutes or more, squeezing using a filter press, and suction filtration using Nutsche.

[0009] Live yeast cells contain intracellular components such as enzymes and proteins, amino acid components, carbohydrate components, and nucleic acid components that are soluble in water or polar solvents, and these components are introduced into the yeast cells. Can inhibit the inclusion of non-lipid soluble substances. Therefore, in order to encapsulate the non-lipid-soluble substance in the yeast cells in a larger amount, it is advisable to use the yeast residue after releasing these intracellular components outside the cells by an enzyme treatment method in advance.
As the enzyme treatment method, any known method can be used, and examples thereof include Babayan, TL and Bezruk.
ov, MG, Acta Biotechnol. 0, 5, 129-136 (1985).
In addition, treatment with a protease or a treatment in which at least one enzyme selected from the group consisting of nuclease, β-glucanase, esterase and lipase is combined with a protease may be performed. Examples of proteases include Alcalase, Neutrase (Novo Corporation), Protease A, M, N, etc., Papain, Neulase F (Amano Corporation), and the like.
Specifically, an aqueous dispersion of yeast cells having an autolyzing enzyme or an aqueous dispersion of yeast cells to which the above-described enzyme has been added is prepared at 30 to 60 ° C. for 1 to 48 hours, preferably 40 to 40 hours.
By incubating at ５０50 ° C. for 15 to 24 hours, the yeast cells can be treated with an enzyme. The aqueous dispersion of yeast cells treated with the enzyme may be separated into a supernatant and yeast residues by centrifugation or the like, and the yeast residues may be used.
Generally, when centrifuged at 4500 rpm for 15 minutes,
The solid content of the yeast cell residue thus treated is about 1
9-21% with about 79-81% moisture.

[0010] Before the enzyme treatment of yeast, a pretreatment may be carried out with a high-pressure homogenizer or the like in order to carry out the enzyme treatment promptly. Specifically, the pretreatment can be performed by dispersing under a pressure of 100 to 600 kg / cm ² using a high-pressure homogenizer. What processed the yeast cell residue which carried out the enzyme treatment as mentioned above with an acidic aqueous solution is more suitable for implementing this invention. Such a treatment reduces the negative charge on the yeast surface and increases the permeability of the substance to be encapsulated into the yeast cells. In particular,
After the enzyme treatment, the yeast residue is suspended in an acidic aqueous solution, and if necessary, adjusted to an appropriate acid concentration, and then the suspension may be heated and stirred for a predetermined time. As the acidic aqueous solution, an aqueous solution of at least one acid selected from the group consisting of hydrochloric acid, phosphoric acid, sulfuric acid, lactic acid, citric acid, acetic acid, ascorbic acid, and the like can be used, but is not particularly limited. The pH of the acidic aqueous solution is suitably 2.0 or less.
1 is preferable, and 0 to 0.5 is more preferable. The yeast residue may be suspended in an acidic aqueous solution so as to have a solid content of 1 to 10%, preferably 2 to 5%. The heating temperature and time of the suspension are preferably set depending on the pH and ionic strength of the system.
50 ° C when treated with 0.5 acidic aqueous solution
The heating is performed at a temperature of not less than 100 ° C and preferably not less than 85 ° C and not more than 100 ° C for 5 minutes to 1 hour, preferably 10 minutes to 30 minutes. At this time, the pH is 0 to 0.5
If a long-term heat treatment of 1 hour or more is carried out with an acidic aqueous solution of the above or a treatment with an excessively acidic aqueous solution having a pH of 0 or less, the strength of the yeast cell wall is reduced accordingly, and the yield of the acid-treated yeast is reduced. The rate may decrease. In the treatment with the acidic aqueous solution as described above, various organic solvents, dispersants, and preservatives can be added as necessary. As an organic solvent, various alcohols such as methanol and ethanol, acetone, hexane and the like, and as a dispersant, sucrose ester, glycerin ester and the like,
As a preservative, benzoic acid, sorbic acid, salicylic acid and the like can be used alone or in combination.

The yeast cell suspension treated with the acidic aqueous solution as described above may be separated into a supernatant and a yeast residue by centrifugation or the like, and the yeast residue may be used. Generally, 450
When centrifuged at 0 rpm for 15 minutes, the solid content of the yeast cell residue thus treated is about 9 to 11% and the water content is about 89 to 91%. The cell wall of the yeast cells obtained by performing the enzyme treatment and the treatment with the acidic aqueous solution as described above is a physically and chemically relatively strong film composed of a glucan, mannan, and chitin layer. A larger amount of non-lipid soluble substance can be encapsulated without impairing the protective function of the substance included in the capsule.

The non-lipid-soluble substance referred to in the present invention means that a functional group which easily forms a bond with a water molecule, for example, a hydroxyl group, a carboxyl group, an amino group, a ketone group, a sulfo group or the like is part of the molecular structure. A relatively low-molecular substance having, preferably, a substance having a solubility of 5 g / 100 ml or more in water, an acidic aqueous solution or an alkaline aqueous solution, and a substance which is solid at room temperature, for example, an amino acid, an organic acid, There are sugars and the like. Specific amino acids include proline, threonine, lysine, arginine, methionine, and the like, and metal salts and esters thereof, and peptides to which these amino acids are bound. Next, as organic acids, malic acid, citric acid, carminic acid, gluconic acid,
Examples thereof include metal salts and esters thereof. Also,
A phosphoric acid metal salt or ester may be used. Examples of the saccharide include maltose, glucose, trehalose and the like. Other examples include inorganic salts such as calcium chloride and sodium chloride. The above is a typical example, and it is needless to say that the present invention is not limited thereto.

The above-mentioned non-lipid-soluble substances are used alone or in combination, and if necessary, mixed with a muddy yeast cell which has been subjected to an enzyme treatment and / or an acid treatment, followed by encapsulation. Specifically, the non-lipid soluble substance may be kneaded together with the muddy yeast cells. The kneading temperature in the encapsulation step is not particularly limited, but is desirably set according to the stability of the non-fat-soluble substance as the core substance. The kneading time may be appropriately set according to the amount of the non-fat-soluble substance to be included, the temperature of the encapsulation step, and the like. Kneading is mechano mill NEW:
Using a mill represented by Okada Seiko Co., Ltd., 100-3
It is good to carry out at a speed of 000 rpm, preferably 100 to 500 rpm. In order to perform the encapsulation advantageously, the yeast intracellular water may be replaced with another liquid before the encapsulation. Since the maximum amount of non-fat-soluble substance that can be included in the yeast depends on its solubility in the water in the yeast, replace the water in the cell with another liquid that dissolves a larger amount of the non-fat-soluble substance to be encapsulated. Thereby, the content of the non-lipid soluble substance in the yeast can be increased.

The yeast cells containing the non-liposoluble substance produced as described above can be recovered by a method such as centrifugation or suction filtration. The recovered yeast cells are
Further, it may be coated with gelatin, zein, alginic acid or the like. In addition, if necessary, washing the yeast,
It is also possible to incorporate operations such as dehydration and adjustment of pH, temperature and pressure. Further, the obtained yeast capsule may be dried by hot air drying, freeze drying, or the like. The microcapsules thus obtained can be used for materials such as food and feed.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to embodiments. Note that the scope of the present invention is not limited to these examples. All yeast weights shown in the examples are actual weights. In addition, the encapsulation rates in yeast cells expressed in% are all percentages by weight.

[Example 1] A muddy yeast obtained by centrifuging a brewer's yeast slurry obtained as a by-product from a beer factory at 4500 rpm for 10 minutes was used as a base material for encapsulation. Proline (solubility:
(167 g / 100 ml), and encapsulation was performed in the following manner.

To 50 g of muddy yeast (solid content: 22.5%), proline was added stepwise from 25 g to 100 g, and kneaded at a rotation speed of 200 rpm for 30 minutes using Mechanomill NEW: Okada Seiko Co., Ltd. However, a phenomenon was observed in which water in the microbial cells was discharged out of the microbial cells throughout, and the sludge cells were again slurried. 25 g to 65 g
In the range, the discharge water amount increased as the addition amount increased, but decreased when the amount exceeded 65 g (● in FIG. 1). In the range of 25 to 65 g, the added proline was observed to be integrated with the cells, but when it exceeded 65 g, it was confirmed that proline was clearly present separately from the cells. . From this, it was presumed that the discharge phenomenon of water out of the cells was compensated for by infiltration of proline into the water inside the cells, that is, the inclusion of proline in the water inside the cells. When the amount of discharged water was at the peak, proline was extracted from the cells obtained by centrifugation and measured, and the encapsulation rate of yeast microcapsules ([proline weight in encapsulated yeast cells / encapsulated yeast] Dry cell weight] × 100 (%)) was 52.6%.

Example 2 A suspension obtained by mixing 400 g of the muddy yeast of Example 1 with 600 g of water as a base material for encapsulation was incubated at 50 ° C. for 17 hours without adjusting the pH. Mud-like autolysed yeast obtained by centrifugation at 4500 rpm for 15 minutes (solid content 20
%) And using proline as the core substance,
As a result, a phenomenon was observed in which the water in the cells was discharged out of the cells and the slurry cells again turned into a slurry (▲ in FIG. 1). When the amount of discharged water was at the peak, proline was extracted from the cells obtained by centrifugation and measured. As a result, the encapsulation rate of the yeast microcapsules was 64.3%.

Example 3 A suspension was prepared by using the muddy autolysed yeast of Example 2 as a base material for encapsulation so that the solid content was 5% and the final hydrochloric acid concentration was 1N. 1
After treatment for 0 minutes, the same treatment as in Example 2 was performed using mud-like acid-treated yeast (solid content: 10%) obtained by centrifugation at 4500 rpm for 15 minutes, using proline as a core substance. As a result, a phenomenon was observed in which the water in the cells was discharged out of the cells throughout the whole, and the sludge cells turned into a slurry. Further, the water discharge phenomenon in this embodiment is the same as that in the first and second embodiments.
At a more drastic level as compared with the water discharge phenomenon at (Fig. 1). When the amount of discharged water was at the peak, proline was extracted from the cells obtained by centrifugation and measured. As a result, the encapsulation rate of the yeast microcapsules was 78.1%.

Example 4 Mud acid-treated yeast 5 of Example 3
0 g (solid content 10%) and carminic acid (purple powder at normal temperature, showing red color when dissolved in water. Solubility: 80 g / 100
The operation of kneading 32 g of water-insoluble in ethanol with ml water, centrifuging, and washing the collected yeast slurry with ethanol and centrifuging to obtain a muddy yeast is repeated three times in the following manner. The cells were encapsulated and the carminic acid attached to the outside of the yeast was physically separated from the yeast cells. Observation under a microscope of the thus obtained product revealed that the inside of the cells was stained red, and it was confirmed that a core substance was present. At this time, yeast microcapsules having an encapsulation rate of 77.8% were obtained.

Example 5 Yeast microcapsules were prepared in the same manner as in Example 3, except that lysine hydrochloride and arginine hydrochloride were used as core substances. At this time, when lysine hydrochloride is used as the core substance, the encapsulation rate is 55.8.
% Of yeast microcapsules, and when arginine hydrochloride was used as the core substance, yeast microcapsules having an encapsulation rate of 60.2% were obtained.

Example 6 In the same manner as in Example 3, yeast microcapsules in which the core substance was methionine (solubility: 3 g / 100 ml of water) were produced. At this time, yeast microcapsules having an encapsulation rate of 8.7% were obtained.

Example 7 After obtaining muddy acid-treated yeast (solid content: 10%) as in Example 3, 100 g of yeast cells
A 5N hydrochloric acid solution was mixed at a rate of 300 g / ml, and the suspension was kept suspended for 3 days to reduce the intracellular water content by 5 g.
After sufficient replacement with N hydrochloric acid, the mixture was centrifuged at 4500 rpm for 15 minutes to obtain a muddy acid-treated yeast in which the intracellular water was replaced with 5N hydrochloric acid. 50 g of muddy acid-treated yeast obtained by the above operation and 36 g of methionine (solubility: 8
(0 g / 100 ml / 5N hydrochloric acid) was directly kneaded to prepare yeast microcapsules containing methionine. At this time, yeast microcapsules having an encapsulation rate of 55.8% were obtained.

Example 8 Yeast microcapsules were produced in the same manner as in Example 3, except that malic acid, anhydrous citric acid and sodium gluconate were used as core substances. At this time, when malic acid was used as the core substance, yeast microcapsules having an encapsulation rate of 83.2% were obtained, and when citric anhydride was used as the core substance, the encapsulation rate was 8%.
5.6% of yeast microcapsules were obtained, and when sodium gluconate was used as the core substance, 71.3% of yeast microcapsules were obtained.

Example 9 Maltose was added to 50 g of the muddy yeast of Example 1 and kneaded. When the amount of addition was 20 to 36 g, the added maltose was integrated with the cells. However, the water discharge phenomenon uniformly observed in the compounds having a carboxyl group tried so far was not confirmed. In addition, when it exceeded 36 g, it was confirmed that maltose was present separately from the cells. When the amount of maltose added was 36 g, yeast microcapsules having a calculated encapsulation rate of 87.8% were obtained.

Example 10 Proof that yeast microcapsules contained a core substance was performed as follows. First, using a French press, each pressure (0, 20
(0, 1000, 1200 and 1500 atm), 50 g of muddy acid-treated yeast cells and 30 g of maltose were directly kneaded. Next, 200 g of water was added to 16 g of the kneaded sample.
Then, the supernatant was sampled at each time, and the amount of maltose contained therein was measured. As a result, as shown in FIG. 2, the non-treated with a French press showed a sustained release of the core substance.
No sustained release was observed in the yeast cells treated at 2,000 atmospheres or more. From these facts, it was found that the cells obtained in Example 9 contained maltose added externally.

Example 11 A yeast microcapsule was prepared in the same manner as in Example 3, except that the core substance was trehalose or sodium chloride. Further, yeast microcapsules were prepared in the same manner as in Example 7 when the core material was 5'-GMP. At this time, when trehalose is used as a core substance, yeast microcapsules having an encapsulation rate of 87.9% are obtained. When sodium chloride is used as a core substance, yeast microcapsules having an encapsulation rate of 72.2% are calculated. Capsules were obtained, and when 5'-GMP was used as the core substance, yeast microcapsules having an encapsulation rate of 84.8% were obtained.

[0028]

The yeast microcapsules of the present invention are useful in the following points. 1) When the yeast and the core substance are simply mixed, the core substance comes into direct contact with the external environment, so that the enzyme modification, light,
Although denaturation due to heat or the like is likely to occur, encapsulation of the core substance with yeast makes it possible to completely block such external environmental stress. 2) The yeast microcapsules obtained by the present invention are granulated,
In terms of processing characteristics such as drying and the like (plasticity and moldability), they are superior to ordinary wax-coated capsules and the like, and are therefore easy to manufacture.

[Brief description of the drawings]

FIG. 1 shows the amount of water discharged when 50 g of yeast residue and various amounts of proline are kneaded.

FIG. 2 shows the release of maltose in acid-treated yeasts treated at each pressure.

Claims (13)

[Claims] 1. A method for producing microcapsules, comprising kneading muddy yeast cells and a non-lipid-soluble substance. 2. The method according to claim 1, wherein the muddy yeast cells are obtained by removing water outside the yeast cells from the yeast cell slurry. 3. The means for removing water outside the yeast cells is one of centrifugation, squeezing and suction filtration.
The manufacturing method as described. 4. The non-lipid soluble substance has a solubility of 5.0 g or more in 100 ml of water, an acidic aqueous solution or an alkaline aqueous solution, and is a solid at room temperature.
The production method according to any one of 5. The yeast cell is one of a live yeast cell, a cell residue obtained by solubilizing and removing intracellular components from the live yeast cell, and a cell obtained by treating the cell residue with an acidic aqueous solution. The production method according to any one of claims 1 to 4. 6. The method according to claim 1, wherein the kneading of the muddy yeast cells and the non-lipid-soluble substance is performed by a shearing force of 100 rpm or more.
6. The production method according to any one of items 5 to 5. 7. The method according to claim 1, wherein the water in the yeast cells is replaced with another liquid before kneading the muddy yeast cells and the non-lipid-soluble substance.
7. The production method according to any one of items 6 to 6. 8. The method according to claim 7, wherein the other liquid is a liquid having a higher solubility for non-lipid soluble substances than water. 9. A microcapsule produced by the method according to claim 1. 10. Microcapsules containing 10% by weight or more of a non-fat-soluble substance added externally inside yeast cells. 11. The microcapsule according to claim 10, wherein the non-lipid-soluble substance has a solubility of 5.0 g or more in 100 ml of water, an acidic aqueous solution or an alkaline aqueous solution, and is a solid at room temperature. 12. The method according to claim 12, wherein the yeast cells are live yeast cells, cell residues obtained by solubilizing and removing intracellular components from the live yeast cells, or those obtained by treating the cell residues with an acidic aqueous solution. 10. The microcapsule according to 10 or 11. 13. The microcapsule according to claim 10, which can be produced by the method according to any one of claims 1 to 8.