JPH08243378A - Prodution of microcapsule - Google Patents

Abstract

PURPOSE: To encapsulate a large quantity of a hydrophobic liquid into a yeast cell in a high ratio by treating the yeast with an enzyme to release components in the cell outside the cell and after that, treating the yeast cell with an acidic aq. solution and encapsulating material to be encapsulated into the yeast cell. CONSTITUTION: A microcapsule is produced by treating the yeast with the enzyme to release the components in the cell outside the cell and after that, treating with the acidic aq. solution and encapsulating the material to be encapsulated into the yeast cell. As the material to be encapsulated, the hydrophobic material, which is a derived lipid selected from among long chain hydrocarbons, long chain alcohols, long chain aminoalcohols, long chain aldehydes, long chain ketones, long chain acids and the salts, terpenoids, steroids, carotenoids or a simple lipid selected from waxes, glycerides, ether glycerides or the like, or a hydrophilic material selected from among amino acids, water soluble vitamin or the like is mentioned.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing microcapsules, and more particularly to a method for producing microcapsules containing a substance to be encapsulated in yeast cells.

[0002]

2. Description of the Related Art A microcapsule contains a liquid, a solid, or a gas, and its circumference is uniformly covered with a thin film of 1 μm.
Micro-capsules, which are fine particles with a size of up to several hundreds of μm, are currently industrially commercialized, including colorless and colored dyes, pharmaceuticals, agricultural chemicals, fragrances, feed materials and food materials. By forming a thin film on the outside of a substance having a certain characteristic, the microcapsule can also contain the characteristic at the same time, and the contained substance can be taken out when necessary. As a method for producing microcapsules, coacervation method, interfacial polymerization method, in situ method and the like are known as promising methods.

On the other hand, some microcapsules using microorganisms have been proposed so far, which are completely different in their manufacturing methods. The microbial microcapsules utilize the cell walls of microorganisms as a membrane material, and thus can be obtained by encapsulating a substance to be encapsulated in a membrane material that has already been produced. Specific examples of the manufacturing method include the following.

In US Pat. No. 4,001,480, a method of culturing a fungus in a medium composition of low nitrogen and high carbon, increasing its lipid content up to 40-60 wt% and encapsulating a substance soluble in the lipid. Has been introduced. According to this method, encapsulation is taken up intracellularly by contact of the substance to be encapsulated with fungi and is held immobile in the fat globules formed intracellularly.

Further, Japanese Patent Laid-Open No. 58-107189 discloses a method for increasing the lipid content of a growth microorganism, which is a growth microorganism having a lipid content of 10 wt% or more recovered from a medium (for example, oil-fat forming yeast, brewer's yeast, etc.). Soluble in these lipid-increasing organic substances after including a liquid selected from lipid-increasing organic substances (eg, aliphatic alcohols, esters, aromatic hydrocarbons, hydrogenated aromatic hydrocarbons) Disclosed is a microbial capsule in which a liquid to be a core substance is encapsulated.

Further, JP-A-61-88871 is characterized in that a fungus having a lipid content of less than 10 wt% contains a hydrophobic substance or a hydrophilic substance, and it is destroyed by applying pressure when necessary to take out the contents. The microbial capsules are listed.

On the other hand, recently, as a simple and excellent method, there has been disclosed a method of using the cell wall of yeast in which intracellular components have been eluted in advance as a base material for microcapsules (Japanese Patent Laid-Open No. Hei 4 (1998) -264242).
-4033, JP-A-4-63127, JP-A-4-117
245, JP-A-5-95791, and JP-A-5-13801.
0, and JP-A-5-253464). However, even by these methods, in order to contain the hydrophobic liquid, which is the core substance, in the cell wall of yeast at a high density, it is necessary to use an emulsifier, which is not the target substance for encapsulation, in combination with the components other than the core substance. It was unavoidable (Japanese Patent Application Laid-Open No. 4-4033, No. 4-63127, No. 4-117245, and No. 5-138010).
Further, as a method which does not necessarily require an encapsulation auxiliary agent such as an emulsifier, a method of treating the cell wall of yeast with an alkaline solution (JP-A-4-63127) or p at the time of encapsulation
A method in which a hydrophobic liquid for encapsulating H after adjusting to a neutral region and a cell wall of yeast are mixed and encapsulation treatment is disclosed (JP-A-5-253464) is disclosed, but the content of fatty acid in the microcapsules is 60. It remained at about%. In addition, compared to fatty acids, triglycerides are easier to use as food additives, but all of the above methods are very big problems when applied to foods, such as exogenous triglycerides are encapsulated in only about 3%. There were many points, and there were many points that had to be improved.

Many hydrophobic liquids containing fats and oils have a nutritional function, and they are generally obtained from animals and plants, but recently, they have also been produced from microorganisms. Some of these hydrophobic liquids are effectively used as food materials and feed materials, but many of them are easily altered by heat, light, oxidants, etc., and if they are liquid, they are very difficult to handle. have. It is expected that if the useful hydrophobic liquid can be encapsulated at a higher density than in the conventional method, these drawbacks can be ameliorated and the application to more advanced applications becomes possible.

In microencapsulating a hydrophobic liquid for application to foods and feeds,
The manufacturing process should be selected with due consideration of the following three points.
First of all, the use of coating materials and encapsulation aids that, when taken as food or feed, should not be of any concern for their safety; secondly, many of the fats and oils undergo thermal denaturation. Due to its susceptibility, avoid encapsulation processes that require conditions of excessively high temperature and long time; thirdly, if possible, make the hydrophobic liquid that is the core substance as high as possible without the need for encapsulation aids. That is, the capsules contained in the density can be prepared. However, at present, a method for producing microcapsules that sufficiently satisfies the above three conditions has not been established.

[0010]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method for manufacturing microcapsules which does not have the above-mentioned drawbacks of the prior art.

[0011]

Therefore, the inventors of the present invention tried various investigations in order to solve the above-mentioned problems, and after enzymatically treating yeast, the intracellular components thereof were released to the outside of the bacterial cells. By treating the yeast cell with an acidic aqueous solution and then encapsulating the substance to be encapsulated in the yeast cell, not only a microcapsule containing a larger amount of a hydrophobic substance but also a microcapsule containing a hydrophilic substance. The inventors have found that it can also be manufactured, and have completed the present invention. That is, the present invention is to release the intracellular components of yeast by treating the yeast with an enzyme, treat the yeast with an acidic aqueous solution, and then encapsulate the substance to be encapsulated in the yeast. The present invention provides a method for producing microcapsules, which is characterized in that

Although not limited to a particular theory, in the method for producing microcapsules of the present invention, the acid treatment on the yeast cell wall surface (negative It is believed that the substance to be encapsulated is more easily taken in, and the electrical repulsion is reduced.

The method for producing microcapsules according to the present invention basically comprises the following steps. Process of releasing intracellular components to the outside of the bacterial cells by enzymatic treatment. Process of treating yeast from which intracellular components have been released with an acidic aqueous solution. Encapsulation process. In addition, yeast washing, dehydration, pH -It is possible to incorporate adjustment of temperature and pressure, and a drying process.
Hereinafter, the present invention will be described in detail according to the above steps.

Yeast is a general term for microorganisms that grow by budding or division, and any yeast may be used in the present invention. Sake yeast, baker's yeast, torrula yeast and the like can be used, and specifically, for example, Saccharomyces cerevisiae of the genus Saccharomyces (Sac
charomyces cerevisiae), Saccharomyces rouxii
(Saccharomyces rouxii), Saccharomyces carlsbergensis, and the genus Candida utilis (Candi
da utilis), Candida trois
picalis), Candida lipol
ytica), Candida flaveri
Etc. can be used. These can be used alone or in combination. The yeast has various shapes depending on the type, but it is preferable that the shape is as close to a spherical shape as possible. The particle size is preferably in the range of 1 to 20 μm. These yeasts used in the present invention may be in a raw state, a dried state, or may be a dead bacterium having no growth ability.

These yeasts used in the present invention have intracellular components such as enzymes and proteins soluble in water or polar solvents, amino acid components, sugars and nucleic acid components, and these components are yeasts. It may inhibit the encapsulation of the substance to be encapsulated in the cells. Therefore, in order to enclose a larger amount of the substance to be encapsulated in the yeast cells, it is necessary to use the yeast residue after releasing these intracellular components to the outside of the cells by the enzyme treatment method in advance. is there. As this enzyme treatment method, any known method can be used, and examples thereof include Babayan, TL and Bezruko.
v, MG, 1Acta Biotechnol. 0, 5, 129-136 (1985)
It is most economical to utilize the autolytic enzyme described in. Besides, 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), Protease A, M, N, etc., papain, Neurase F (Amano) and the like. Specifically, an aqueous dispersion of yeast cells having an autodigestive enzyme or an aqueous dispersion of yeast cells added with the enzyme as described above is kept at 30 to 60 ° C. for 1 to 48 hours, preferably 40 to The yeast cells can be treated with an enzyme by incubating at 50 ° C. for 15 to 24 hours.
An aqueous dispersion of yeast cells treated with the enzyme may be separated into a supernatant and a yeast residue by centrifugation or the like, and the following treatment may be performed using the yeast residue.

For the purpose of rapid enzymatic treatment, yeast may be pretreated with a high-pressure homogenizer or the like before the enzymatic treatment. Specifically, pretreatment can be performed by preliminarily dispersing with a high-pressure homogenizer under a pressure of 100 to 600 kg / cm ² , and then dispersing under a pressure of 800 to 2000 kg / cm ² .

Next, the enzyme-treated yeast cells are treated with an acidic aqueous solution. This treatment reduces the negative charge on the yeast surface and increases the permeability of the substance to be encapsulated into the yeast cells. Specifically, the yeast residue after the enzyme treatment may be suspended in an acidic aqueous solution and, if desired, adjusted to an appropriate acid concentration, and then the suspension may be heated and stirred for a predetermined time. As the acidic aqueous solution used in the method of the present invention,
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, preferably 0 to 1, and more preferably 0 to 0.5. Further, the yeast residue has a solid content concentration of 1 to 10%, preferably 2 in an acidic aqueous solution.
It is recommended to suspend the solution so that the concentration becomes ~ 5%. The heating temperature and time of this suspension are preferably set depending on the pH and ionic strength of the system, but for example, the pH is 0 to 0.5.
50 ° C or more when treated with the acidic aqueous solution of
It is preferable to heat at a temperature of 00 ° C. or lower, preferably 85 ° C. or higher and 100 ° C. or lower, for 5 minutes or longer and 1 hour or shorter, preferably 10 minutes or longer and 30 minutes or shorter. At this time, if the heat treatment is carried out for a long time of 1 hour or more with an acidic aqueous solution having a pH of 0 to 0.5, or if the treatment is carried out with an excessively acidic aqueous solution having a pH of 0 or less, the strength of the yeast cell wall is accordingly In some cases, resulting in a decrease in the yield of acid-treated yeast. In the treatment with the acidic aqueous solution as described above, various organic solvents, dispersants, and preservatives can be added as necessary. As the organic solvent, various alcohols such as methanol and ethanol, acetone, hexane and the like, as the dispersant, sucrose ester, glycerin ester and the like, as the preservative, benzoic acid, sorbic acid, salicylic acid, etc. alone, Alternatively, they can be used 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 for the following treatment. The cell wall of the yeast cell 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 the substance to be encapsulated can be encapsulated without impairing the protective function of the substance to be encapsulated. Microcapsules are produced by encapsulating the substance to be encapsulated in the yeast cells treated as described above.

The substance to be encapsulated may be of any nature, hydrophobic, hydrophilic or amphipathic, but the method of the invention encapsulates a hydrophobic substance, in particular a hydrophobic liquid. It is effective for The hydrophobic liquid is a substantially water-insoluble liquid, a liquid which becomes a water-insoluble liquid by heating, and a fat-soluble substance such as fatty acid ester or steroid, which is a suitable liquid (for example, the substances described below. Of which is a liquid) that contains a hydrophobic liquid dissolved in. Specifically, as simple lipids, monoester type chain simple waxes such as palmitic acid methyl ester composed of higher fatty acid and higher alcohol, and cholesterol ester, sitosterol ester,
Ring-containing simple waxes represented by sterol esters such as ergosterol esters and esters of fat-soluble vitamins A, D, E and simple waxes containing cyanolipids, diol lipids, complex waxes such as diesters, monoolein, Monoglycerides and diglycerides such as monostearin, triolein, soybean oil, corn oil, rice bran oil, safflower oil, cottonseed oil, olive oil, castor oil,
Cod oil, squid oil, sardine oil, lard, beef tallow, sheep fat, horse oil,
In addition, triglycerides represented by microbial oils and fats, monoalkyl and dialkyl such as chimyl alcohol, batyl alcohol, monoalkyl monoacyl, monoalkyldiacyl, alkylalkyl ether lipids such as trialkyl type and monoalkenyl, dialkenyl,
Monoalkenyl monoacyl, monoalkenyl diacyl,
Examples include alkenyl glyceryl ether lipids such as trialkenyl type and ceramides. In addition, long-chain hydrocarbons represented by saturated and unsaturated straight-chain, mono-branched and poly-branched long chain hydrocarbons and octacosanol obtained by oxidation of these long chain hydrocarbons, dihydrosphingosine, Long-chain amino alcohols such as sphingosine, phytosphingosine, dehydrophytosphingosine, long-chain aldehydes often found in citronellal, farnesal and insect pheromones, phylloquinone,
Linear ketones such as ubiquinone, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, various fatty acids typified by docosahexaenoic acid, and long-chain acids such as hydroxy acid, keto acid, dicarboxylic acid And its salts, monoterpenes such as hemiterpenes, limonene, menthol, citral and ionone, bisabolen, farnesol, nerolidol, cyperone, hinokinoic acid

Diterpenes such as sesquiterpenes such as campholene, campholene, phytol, hinokiol, sugiol, abietic acid, chlorophyll, retinol, tocopherol, and phylloquinone, and terpenoids such as polyterpenes such as tetraterpenes and menaquinones and ubiquinones, cholesterol, sitosterol. , Ergosterol, bile acid, sex hormone, adrenal lipid hormone, cardiotoxin genin, steroid sapogenin, steroids such as solanidine, and carotenoids such as phytoene, lycopene, carotene, xanthophylls, citraurine, and capsanthin. Furthermore, phosphatidylcholine and phosphatidylethanolamine such as soybean lecithin as a complex lipid, glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, glycerophosphonolipids such as cardiolipin,
Ether glycerophospholipids such as plasmalogen, ceramide phosphoric acid, sphingolipids such as sphingomyelin, phospholipids such as sphingophosphonolipids such as ceramide ciliatin, other glycolipids such as glyceroglycolipids and glycosphingolipids, saponins , Steroid glycosides such as solanin, glycolipids such as fatty acid sugars and lipopolysaccharides, phosphoglycolipids, sulfur lipids, amino acid lipids and the like.

Furthermore, a fat-soluble liquid such as fenitrothion or pyraclofas can be used. Examples of the core substance include emulsifiers represented by glycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, polysorbates and the like. A hydrophilic substance is a relatively low-molecular substance having a functional group that easily forms a bond with a water molecule, that is, a hydroxyl group, a carboxyl group, an amino group, a ketone group, a sulfo group, etc., as part of its molecular structure. , Substantially soluble in water. Specifically, first of all, amino acids can be mentioned. Amino acids such as proline, threonine and lysine, as well as their metal salts, esters and peptides are included. The sugars can then be mentioned. In addition to monosaccharides such as glucose, galactose and fructose, disaccharides such as maltose and sucrose, oligosaccharides such as raffinose, amino sugars and sugar alcohols are included. Next are water-soluble vitamins.
There are B vitamins such as thiamine and riboflavin, ascorbic acid, folic acid and choline. In addition, nucleic acid-related substances can be mentioned. In addition to mononucleotides such as 5'-GMP and 5'-IMP, there are bases, nucleosides, and oligonucleotides.

These substances to be encapsulated, alone or in combination, are mixed with the yeast cells that have been subjected to the above-mentioned treatment, and then encapsulated. Specifically, the substance to be encapsulated is added to an aqueous dispersion of the yeast residue that has been subjected to the above treatment, and the pH of the dispersion is adjusted, if desired, and then stirred for a certain period of time at a certain temperature. It can be done by The ratio of the substance to be encapsulated and the yeast cells (solid content) is appropriately 2: 1 to 1: 4, preferably 1: 1 to 1: 2. The solid concentration of the yeast cells in the dispersion is 1 to 1
0% is suitable, and 2-5% is preferable. If desired
The pH of the dispersion is 5-9, preferably 6.5-7.
Sodium hydroxide, potassium hydroxide,
You may adjust using a pH adjuster, such as heavy sow. The stirring temperature in the encapsulation step is not particularly limited, but is preferably 40 to 80 ° C. Further, the stirring time may be appropriately set according to the amount of the substance to be encapsulated to be encapsulated, the temperature of the encapsulation process, and the like. Stirring may be performed using a homogenizer at a speed of 1000 to 10000 rpm, preferably 2000 to 4000 rpm. Further, although not necessarily required, a surfactant or a hydrophilic organic solvent may be added to help improve the dispersibility of the substance to be encapsulated. Further, if necessary, various deterioration preventing agents such as a hardening agent, a preservative, an antioxidant and the like can be added for encapsulation. This step is completely different from the culture of yeast, and the supply of dissolved oxygen, sugar source,
It is completely unnecessary to add a nutrient source such as a nitrogen source.

The yeast cells containing the substance to be encapsulated, produced as described above, are collected and washed. The yeast cells can be collected by a method such as centrifugation or suction filtration. In addition, you may dry by performing processes, such as washing and hot-air drying, and freeze-drying, as needed. The microcapsules thus obtained can be used as materials such as foods and feeds.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below with reference to Examples. The scope of the present invention is not limited to these examples. All yeast weights given in the examples are dry weights. In addition, all the encapsulation rates in yeast cells expressed in% are% by weight.

[Example 1] <Process for eluting intracellular components by enzyme treatment> 200 g of an aqueous dispersion containing 20 g of brewer's yeast (Saccharomyces cerevisiae) was shaken and cultivated at 50 ° C for 17 hours. The cells were shaken and the components inside the cells were eluted outside the cells by the autodigestion method. After the eluate and the yeast residue were separated by a centrifugation operation, a paste having a solid content of about 20% was obtained as an enzyme-treated yeast residue (hereinafter referred to as “enzyme-treated residue”).

<Treatment of Enzymatic Treatment Residue with Acidic Aqueous Solution>
The above enzyme treatment residue was suspended in a hydrochloric acid solution so that the solid content became 5%, and the final hydrochloric acid concentration was adjusted to 0.05, 0.1,
It was adjusted to 0.5, 1.0, and 2.0 N, respectively. This suspension is heated in a boiling water bath at 85 ° C or higher to 1
The mixture was heated for 0 minutes, centrifuged, and then washed to obtain a hydrochloric acid-treated residue of the enzyme-treated yeast (hereinafter referred to as "hydrochloric acid-treated residue"). The residue treated with hydrochloric acid had a water content of 90 to 85%, that is, a solid content of 10 to 15%.

<Encapsulation Step> Each of the hydrochloric acid-treated residues obtained as described above was suspended in distilled water so that the solid content was 5% and oleic acid or triolein was 7.5%. , PH was adjusted to 7.0 with sodium hydroxide. This suspension was stirred for 16 hours with a homogenizer at a temperature of 70 ° C. Encapsulated yeast cells were obtained by centrifugation. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight was measured, and the encapsulation rate ([weight of lipid in encapsulated yeast cells / dry weight of encapsulated yeast cells] x 10
0 (%)) was calculated. The results are shown in Tables 1 and 2. For example, when oleic acid is encapsulated by 1N hydrochloric acid treatment, the recovery rate of the encapsulated yeast is 62.6% of the total solid content of the hydrochloric acid treatment residue used and oleic acid.
The final recovery of encapsulated oleic acid was 75.5%.

The capsule prepared by the above-mentioned method was adjusted to pH 2,
The suspension was suspended in a solution of 4, 7, and 9 adjusted with hydrochloric acid or sodium hydroxide, left standing for 24 hours, and the amount of lipid remaining in the capsule was measured. Also, after heating at 120 ° C. for 20 minutes, the amount of residual lipid was measured in the same manner, but in any case, no outflow of lipid component was observed. From this, it can be seen that these capsules are extremely stable against changes in pH and temperature.

[Comparative Test 1-1] The procedure of Example 1 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight thereof was measured, and the encapsulation rate was calculated. The encapsulation rate of oleic acid was 36.0% (see Table 1). The untreated group) and the encapsulation rate of triolein were 4.6% (untreated group in Table 2). In addition, Example 1
In, the encapsulation time required to reach this encapsulation rate was within 1 hour, suggesting that the residue treated with hydrochloric acid can be encapsulated extremely quickly as compared with the self-digestion residue.

[0030]

[Table 1] Table 1 Encapsulation rate of oleic acid-encapsulated yeast ───────────────────── Concentration of hydrochloric acid Treatment solution Encapsulation rate (N) pH ¹⁾ (%) ───────────────────── 0.0 (untreated) 5.80 36.0 0.05 1.35 56.2 0.1 1 0.07 54.0 0.5 0.34 69.6 1.0 0.01 72.4 2.0 *** 59.8 ──────────────────── ─── ¹⁾ pH is the measured value

[0031]

[Table 2] Table 2 Encapsulation rate of triolein-encapsulated yeast ───────────────────── Concentration of hydrochloric acid Treatment solution Encapsulation rate (N) pH ¹⁾ (%) ───────────────────── 0.0 (untreated) 5.80 4.6 0.05 1.35 5.2 0.1 1 0.07 5.8 0.5 0.34 31.3 1.0 0.01 25.3 2.0 *** 19.7 ──────────────────── ─── ¹⁾ pH is the measured value

[Comparative Test 1-2] The procedure of Example 1 was repeated except that the elution treatment of the intracellular components by the enzyme treatment was not performed and the treatment with the acidic aqueous solution was performed using a 1N hydrochloric acid solution. Acid or triolein was encapsulated in yeast cells. At this time, the encapsulation rate of oleic acid was 55.3%, and the encapsulation rate of triolein was 7.7%. This indicated that the encapsulation rate was significantly reduced in the absence of enzyme treatment, and triolein was hardly encapsulated.

[Comparative Test 1-3] In the treatment of the enzyme treatment residue with an acidic aqueous solution, treatment with a hydrochloric acid solution was carried out to 0.0
The procedure of Example 1 was repeated except that the treatment with a 1N sodium hydroxide solution (pH = 12.0, optimum conditions according to JP-A-4-63127) was replaced, and oleic acid or triolein was introduced into the yeast cells. Encapsulated. At this time, the encapsulation rate of oleic acid was 46.5%, and the encapsulation rate of triolein was 4.5%. This indicates that the treatment with acid is more effective in the encapsulation than the treatment of the yeast residue with alkali.

[Example 2] <Elution process of intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzymatic Treatment Residue with Acidic Aqueous Solution> The same procedure as in Example 1 was performed, but the final hydrochloric acid concentration was 1N.

<Encapsulation Step> Each of the hydrochloric acid-treated residues obtained above was suspended in distilled water so that the solid content was 5% and soybean oil had a concentration of 7.5%, and the pH was 7.0. Adjusted with sodium hydroxide. This suspension was stirred with a homogenizer for 16 hours under the condition of a temperature of 70 ° C. Encapsulated yeast cells were obtained by centrifugation. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight was measured, and the encapsulation rate was calculated. As a result, the encapsulation rate of soybean oil was 22.0%.

[Comparative Test 2] The procedure of Example 2 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. In the obtained encapsulated yeast cells, the encapsulation rate of soybean oil was significantly reduced to 4.4%. This shows that the method of the present invention in which yeast cells are treated with an acidic aqueous solution after enzymatic treatment can be applied not only to triolein but also to triglycerides in general.

[Example 3] <Process for eluting intracellular components by enzyme treatment> Example 1
Prior to the elution treatment of the intracellular components by self-digestion, the brewer's yeast was predispersed in water at a solid concentration of 10% under a pressure of 500 kg / cm ² using a high-pressure homogenizer, and then 1
Further dispersion was carried out under a pressure of 000 kg / cm ² . Then, the mixture was shaken in a shaking incubator at a temperature of 50 ° C. for 8 hours to elute the water-soluble components in the cells outside the cells by autolysis and separate the eluate and the yeast residue by centrifugation. , An enzyme-treated residue was obtained.

<Treatment of Enzymatic Treatment Residue with Acidic Aqueous Solution>
The same procedure as in Example 1 was repeated except that the final hydrochloric acid concentration was 1N.
And

<Encapsulation Step> Oleic acid or triolein was encapsulated by the same operation as in Example 1.
At this time, the encapsulation rate of oleic acid was 74.6%, and the encapsulation rate of triolein was 18.6%. In addition, these capsules were very stable to pH changes and temperature changes, and even when suspended in water and left standing for 24 hours, no outflow of lipid components was observed.

[Comparative Test 3] The procedure of Example 3 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight was measured, and the encapsulation rate was calculated. The encapsulation rate of oleic acid was 42.7%. The encapsulation rate was 4.6%.

[Example 4] <Elution process of intracellular components by enzyme treatment> Example 1
The same method was used.

<Treatment of Enzymatic Treatment Residue with Acidic Aqueous Solution>
The procedure of Example 1 was repeated, except that hydrochloric acid was replaced with sulfuric acid in the treatment of the enzyme treatment residue with an acidic aqueous solution to give final sulfuric acid concentrations of 0.5 and 1.0 N. At this time, the solid contents of the sulfuric acid treatment residue were 14% and 12%, respectively.

<Encapsulation Step> Each of the sulfuric acid treatment residues obtained as described above was suspended in distilled water so that the solid content was 5% and triolein was 7.5%. Adjusted to 7.0 with sodium hydroxide. This suspension was stirred with a homogenizer for 16 hours under the condition of a temperature of 70 ° C. Encapsulated yeast cells were obtained by centrifugation. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform and the weight was measured to calculate the encapsulation rate. Table 3 shows the results. In addition, these capsules are very stable against changes in pH and temperature, so even if they are suspended in water and left for 24 hours,
No outflow of lipid components was observed.

[Comparative Test 4-1] The procedure of Example 4 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight thereof was measured, and the encapsulation rate was calculated. The encapsulation rate of triolein was 4.6% (Table 3 Untreated group).

[0045]

[Table 3] Table 3 Encapsulation rate of triolein-encapsulated yeast ───────────────────── Sulfuric acid concentration Treatment solution Encapsulation rate (N) pH ¹⁾ (%) ───────────────────── 0.0 (untreated) 5.84 4.6 0.5 0.62 12.7 1.00 .33 34.2 ───────────────────── ¹⁾ pH is the measured value

[Comparative Test 4-2] The trio was repeated by repeating the procedure of Example 4 except that the elution treatment of the intracellular components by the enzyme treatment was not performed and the treatment with the acidic aqueous solution was performed using a 1N sulfuric acid solution. Rain was encapsulated in yeast cells. At this time, the encapsulation rate of triolein was 4.2%. From this, it was revealed that triolein was hardly encapsulated without the enzyme treatment.

[Example 5] <Process for eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzymatic Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed.

<Encapsulation Step> Each of the hydrochloric acid-treated residues obtained as described above was suspended in distilled water to a solid content of 5% and palmitic acid methyl ester to a concentration of 7.5%. The pH was adjusted to 7.0 with sodium hydroxide. This suspension was stirred with a homogenizer for 16 hours under the condition of a temperature of 70 ° C. Encapsulated yeast cells were obtained by centrifugation. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight was measured, and the encapsulation rate was calculated. The encapsulation rate of palmitic acid methyl ester was 54.7%. In addition, these capsules were very stable to pH changes and temperature changes, and even when suspended in water and left standing for 24 hours, no outflow of lipid components was observed.

[Comparative Test 5] The procedure of Example 5 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight was measured, and the encapsulation rate was calculated. The encapsulation rate of palmitic acid methyl ester was 2
It was 5.4%.

[Example 6] <Process for eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzymatic Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed.

<Encapsulation Step> The hydrochloric acid-treated residue obtained above was suspended in distilled water so that the solid content was 5% and oleic acid monoglyceride was 7.5%.
The pH was adjusted to 7.0 with sodium hydroxide. This suspension was mixed with a homogenizer at a temperature of 70 ° C for 1 hour.
Stir for 6 hours. Encapsulated yeast cells were obtained by centrifugation. Extract the lipid component contained in the obtained encapsulated yeast cells with chloroform, measure the weight,
When the encapsulation rate was calculated, the encapsulation rate of oleic acid monoglyceride was 34.0%. In addition, these capsules were very stable to pH changes and temperature changes, and even when suspended in water and left standing for 24 hours, no outflow of lipid components was observed.

[Comparative Test 6] The procedure of Example 6 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight was measured, and the encapsulation rate was calculated. The encapsulation rate of oleic acid monoglyceride was 11.9.
%Met.

[Example 7] <Process for eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzymatic Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed.

<Encapsulation Step> The hydrochloric acid-treated residue obtained above was suspended in distilled water so that the solid content was 5% and soybean lecithin (phosphatidylcholine) was 7.5%, and the pH was adjusted to 7. Adjusted to 0 with sodium hydroxide. This suspension was stirred with a homogenizer for 16 hours under the condition of a temperature of 70 ° C. Encapsulated yeast cells were obtained by centrifugation. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight was measured, and the encapsulation rate was calculated. As a result, the encapsulation rate of soybean lecithin was 31.3%. Also,
These capsules were very stable to pH changes and temperature changes, and even when suspended in water and left standing for 24 hours, no outflow of lipid components was observed.

[Comparative Test 7] The procedure of Example 7 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the weight was measured, and the encapsulation rate was calculated. As a result, the encapsulation rate of soybean lecithin was 6.41%.

[Embodiment 8] <Process for eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzymatic Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed.

<Encapsulation Step> The hydrochloric acid-treated residue obtained above was 5% in solid content, and a 30% retinyl palmitate solution (in corn oil) was diluted with distilled water to a concentration of 7.5%. It was suspended and the pH was adjusted to 7.0 with sodium hydroxide. This suspension was stirred with a homogenizer for 16 hours under the condition of a temperature of 70 ° C. Encapsulated yeast cells were obtained by centrifugation. When the lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform and the content of retinyl palmitate was measured by HPLC, the encapsulation rate of retinyl palmitate was 6.70%. Also, these capsules have a pH
It is very stable against changes in temperature and temperature, and is suspended in water. 2
Even after standing for 4 hours, no outflow of lipid components was observed.

[Comparative Test 8] The procedure of Example 8 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, the content was measured, and the encapsulation rate was calculated. The encapsulation rate of retinyl palmitate was 3.11%.
Met.

[Example 9] <Process for eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzyme Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed. <Encapsulation process> The hydrochloric acid treatment residue obtained above was 3% in solid content, and lecithin was 31% and cephalin was 3%.
A phospholipid mixture containing 2% ("Rion PK" manufactured by Riken Vitamin Co.) was suspended in distilled water to a concentration of 5%, and p
The H was adjusted to 7.0 with sodium hydroxide. This suspension was placed in a homogenizer at a temperature of 70 ° C. for 16 minutes.
Stirred for hours. Encapsulated yeast cells were obtained by centrifugation. The lipid component contained in the obtained encapsulated yeast cells was extracted with chloroform, and the lecithin and cephalin contents therein were measured by HPLC, and the total was 16.9%. Also, these capsules are
Except for some leakage of inclusion compound under H2 condition, it is very stable to pH change and temperature change, and even if suspended in water and left for 24 hours, no outflow of lipid component was observed. It was

[Comparative Test 9] Encapsulated yeast cells were obtained by repeating the procedure of Example 9 except that the residue treated with the enzyme was not treated with the acidic aqueous solution. The lipid components contained in the obtained encapsulated yeast cells were extracted with chloroform, and the lecithin and cephalin contents therein were measured. The total was 1.02%.

[Example 10] <Process for eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzyme Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed. <Encapsulation Step> The hydrochloric acid-treated residue obtained as described above is added to a solid content of 5%, and 9 kinds of amino acids, ie, alanine, valine, proline, serine, threonine, cysteine, glycine, arginine, and lysine are added to a concentration of 1%. The resulting suspension was suspended in distilled water and adjusted to pH 7.0 with sodium hydroxide. This suspension was stirred for 16 hours with a homogenizer under the condition of a temperature of 30 ° C. Encapsulated yeast cells were obtained by centrifugation. Amino acids contained in the obtained encapsulated yeast cells were extracted with a 50% ethanol solution and measured by an amino acid analyzer.
The total encapsulation rate of 9 amino acids was 4.86%.

[Comparative Test 10-1] The procedure of Example 10 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. Amino acids contained in the obtained encapsulated yeast cells were extracted with a 50% ethanol solution and measured by an amino acid analyzer, and the encapsulation rate of 9 kinds of amino acids was 0.83% in total.
Met. [Comparative Test 10-2] Neither the elution treatment of the intracellular components by the enzyme treatment nor the treatment with the acidic aqueous solution was performed, and only the encapsulation step was performed according to Example 10 to obtain the encapsulated yeast cells. When the amino acids contained in the obtained encapsulated yeast cells were extracted with a 50% ethanol solution and measured by an amino acid analyzer, the encapsulation rate of 9 kinds of amino acids was 1.43% in total.

[Embodiment 11] <Process of eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzyme Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed. <Encapsulation Step> The hydrochloric acid-treated residue obtained above was suspended in distilled water so that the solid content was 5% and proline was 5%, and the pH was adjusted to 7.0 with sodium hydroxide. This suspension was placed under the condition of a temperature of 30 ° C.
The mixture was stirred with a homogenizer for 16 hours. Encapsulated yeast cells were obtained by centrifugation. When the proline contained in the obtained encapsulated yeast cells was extracted with a 50% ethanol solution and measured by an amino acid analyzer, the encapsulation rate of proline was 2.80% (the acid-treated yeast group in FIG. 1). , Wash 0 times). The capsules were washed by resuspending them in a large amount of water and then collecting them by centrifugation. As a result, a significant amount of proline was retained in the yeast cells after repeated washing 4 times. Was found (the acid-treated yeast group in FIG. 1, washed 4 times).

[Comparative Test 11] Encapsulated yeast cells were obtained by repeating the procedure of Example 11 except that the enzyme treatment residue was not treated with an acidic aqueous solution. When the proline contained in the obtained encapsulated yeast cells was extracted with a 50% ethanol solution and measured by an amino acid analyzer, the encapsulation rate was 1.29% (self-digesting yeast group in FIG. 1, washing 0 Times). Further, when this capsule was washed in the same manner as in Example 11, almost no proline was retained in the yeast cells at the end of washing twice (autolysis digested yeast group in FIG. 1, washed). 2 times).

[Embodiment 12] <Elution process of intracellular components by enzyme treatment> Embodiment 1
The same method was used. <Treatment of Enzyme Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed. <Encapsulation Step> The hydrochloric acid-treated residue obtained above was suspended in distilled water so that the solid content was 5% and threonine was 5%, and the pH was adjusted to 7.0 with sodium hydroxide. This suspension was stirred for 16 hours with a homogenizer under the condition of a temperature of 30 ° C. Encapsulated yeast cells were obtained by centrifugation. Threonine contained in the obtained encapsulated yeast cells was extracted with a 50% ethanol solution and measured by an amino acid analyzer.
The encapsulation rate of threonine was 0.50% (Fig. 2
Acid-treated yeast group, washed 0 times). When this capsule was washed twice by resuspending it in a large amount of water and then collecting it by centrifugation, the content of threonine retained in the yeast cells was reduced by only 23% (Fig. 2).
Acid-treated yeast group, twice washed).

[Comparative Test 12] The procedure of Example 12 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. When threonine contained in the obtained encapsulated yeast cells was extracted with a 50% ethanol solution and measured by an amino acid analyzer, the encapsulation rate was 0.53% (self-digesting yeast group in FIG. 2, washing 0 Times) and the level was almost the same as in Example 12. However, when this capsule was washed twice by the same method as in Example 12, the threonine content retained in the yeast cells was significantly reduced (autodigested yeast group in FIG. 2, twice washing).

[Example 13] <Process for eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzyme Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed. <Encapsulation step> The hydrochloric acid-treated residue obtained above was suspended in distilled water so that the solid content was 5% and ascorbic acid was 5%, and the pH was adjusted to 7.0 with sodium hydroxide. . This suspension was stirred for 16 hours with a homogenizer under the condition of a temperature of 30 ° C. Encapsulated yeast cells were obtained by centrifugation. Ascorbic acid contained in the obtained encapsulated yeast cells was extracted with metaphosphoric acid and measured with an F-kit (manufactured by Boehringer Mannheim), and the encapsulation rate of ascorbic acid was 1.17% ( Ascorbic acid in FIG. 3, acid-treated yeast group).

[Comparative Test 13] The procedure of Example 13 was repeated except that the residue treated with the enzyme was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. Ascorbic acid contained in the obtained encapsulated yeast cells was extracted with metaphosphoric acid and measured by F-kit (Boehringer Mannheim), and the encapsulation rate was 0.43%.
(Ascorbic acid in FIG. 3, self-digesting yeast group).

[Example 14] <Process for eluting intracellular components by enzyme treatment> Example 1
The same method was used. <Treatment of Enzyme Treatment Residue with Acidic Aqueous Solution> The same method as in Example 1 was performed. <Encapsulation Step> The hydrochloric acid-treated residue obtained above was suspended in distilled water so that the solid content was 5%, and 5 ′ guanylic acid monophosphate (hereinafter 5′-GMP) was 5% in concentration. The pH was adjusted to 7.0 with sodium hydroxide. This suspension was stirred for 16 hours with a homogenizer under the condition of a temperature of 30 ° C. Encapsulated yeast cells were obtained by centrifugation. 5'-GMP contained in the obtained encapsulated yeast cells was extracted with 80% ethanol,
When measured by HPLC, the encapsulation rate of 5′-GMP was 1.13% (5′-GMP in FIG. 3, acid-treated yeast group).

[Comparative Test 14] The procedure of Example 14 was repeated except that the enzyme treatment residue was not treated with an acidic aqueous solution to obtain encapsulated yeast cells. When 5'-GMP contained in the obtained encapsulated yeast cells was extracted with 80% ethanol and measured by HPLC, it was found that the 5'-GMP was hardly encapsulated (5'-G in FIG. 3).
MP, autolyzed yeast group).

[0071]

Industrial Applicability According to the method of the present invention, it becomes possible to enclose a large amount of hydrophobic liquid in yeast cells in a short time at a high ratio (encapsulation rate). The hydrophobic liquid component yeast capsule produced by the method of the present invention has good retention and stability, and is practically sufficient for use.
Surprisingly, the method of the present invention could easily realize encapsulation of triglycerides, fatty acid esters, and fat-soluble vitamins, which had been conventionally difficult. Furthermore, the method of the present invention makes it possible to enclose a larger amount of a hydrophilic substance in yeast cells at a higher ratio as compared with the conventional encapsulation method. The hydrophilic component yeast capsule produced by the method of the present invention has good retention and stability. As described above, the method of the present invention is a microencapsulation method using microorganisms and is a means excellent in terms of quality and industry.

[0072]

[Brief description of drawings]

FIG. 1 shows the encapsulation rate of proline-encapsulated yeast after 0, 2 and 4 washings.

FIG. 2 shows the encapsulation rate of threonine-encapsulated yeast after 0 and 2 washings.

FIG. 3 Ascorbic acid encapsulated yeast and 5′-G
The encapsulation rate of MP encapsulated yeast is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area // (C12N 1/16 C12R 1: 865)

Claims (16)

[Claims] 1. A yeast is enzymatically treated to release its intracellular components to the outside of the bacterial body, the yeast bacterial body is treated with an acidic aqueous solution, and then the substance to be encapsulated is encapsulated in the yeast bacterial body. A method for producing a microcapsule, which is characterized in that 2. The method according to claim 1, wherein the substance to be encapsulated is a hydrophobic substance. 3. The method according to claim 2, wherein the hydrophobic substance is a liquid. 4. The method of claim 3, wherein the hydrophobic liquid is a lipid. 5. The lipid is at least selected from the group consisting of long chain hydrocarbons, long chain alcohols, long chain amino alcohols, long chain aldehydes, long chain ketones, long chain acids and salts thereof, terpenoids, steroids and carotenoids. The method according to claim 4, which is a kind of derivative lipid. 6. The method according to claim 4, wherein the lipid is at least one simple lipid selected from the group consisting of wax, glyceride, ether glyceride and ceramide. 7. The method according to claim 4, wherein the lipid is at least one complex lipid selected from the group consisting of phospholipids, glycolipids, phosphoglycolipids, sulfur lipids and amino acid lipids. 8. The method according to claim 1, wherein the substance to be encapsulated is a hydrophilic substance. 9. The method according to claim 8, wherein the hydrophilic substance is at least one substance selected from the group consisting of amino acids, water-soluble vitamins, nucleotides and sugars. 10. The method according to claim 1, wherein the enzyme is a protease. 11. The method according to claim 1, wherein the enzyme is a combination of at least one enzyme selected from the group consisting of nuclease, β-glucanase, esterase and lipase with a protease. 12. The method according to claim 1, wherein the enzyme is a self-digesting enzyme possessed by yeast. 13. The method according to claim 1, wherein the acidic aqueous solution has a pH of 2.0 or less. 14. The method according to claim 13, wherein the acidic aqueous solution has a pH of 0 to 0.5. 15. The aqueous acid solution is an aqueous solution of at least one acid selected from the group consisting of hydrochloric acid, phosphoric acid, sulfuric acid, citric acid, acetic acid, ascorbic acid, and lactic acid.
The described method. 16. The yeast is Saccharomyces cerevisiae, Saccharomyces
Ruki (Saccharomyces rouxii), Saccharomyces
Carlsbergensis (Saccharomyces carlsbergensis
i), Candida utilis, Candida tropicalis, Candida lipolytica, and at least one selected from the group consisting of Candida flaveri. It is a microorganism.
The described method.