JPH07289885A - Microcapsulated yeast, its production and feed containing the same - Google Patents

Abstract

PURPOSE:To obtain a yeast encapsulated with phospholipids which is highly practical by bringing a yeast into contact with a soln. containing univalent alcohol having specified carbon number and containing phospholipids. CONSTITUTION:This yeast encapsulated with phospholipids is obtd. by bringing the yeast into contact with an alcohol soln. containing phospholipids. As for alcohol, univalent alcohol having 1-4 carbon number, preferably that having 1-3 carbon number is used. Ethanol is most preferable. As for the concn. of alcohol in the system, it is preferable to carry out the process generally in about >=50wt.% concn. of alcohol. The amt. of the phospholipids used is not specified but is determined according to the amt. to be encapsulated, the amt. of solvent used or other conditions. Generally, about 5-100wt.% phospholipids based on the yeast (dry weight) is enough, and if necessary, about >=100wt.% may be used. The density of yeast cells (dry weight) in the suspension liquid is not specified but is about 5-20wt.%.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microcapsule in which a yeast is used as a microcapsule film and a lipid is included therein. By encapsulating the lipid in the yeast, it is possible to improve the deterioration due to light, heat, an oxidizing agent and the like and the handling difficulty. Also, by encapsulating a dye together with a lipid, it can be used as a pressure-sensitive biocapsule for a recording material,
By encapsulating a specific drug together with the lipid, it can be used as an insecticide or pesticide. In particular, by encapsulating phospholipids, which have the effect of improving the absorbability of various compounds in the body as lipids, it is possible to improve the absorbency of useful substances produced in yeast cells and the absorbency of active substances included with phospholipids. It can be widely used in the fields of medicine, food, feed, etc. The use of a yeast that is not toxic to the living body as a microcapsule film is particularly convenient in terms of utilization in the fields of medicine, food, feed and the like. Thus, by encapsulating lipids, it is expected to be applicable to various advanced uses.

[0002]

2. Description of the Related Art Microcapsules contain liquids, solids, and gases as fine particles having a size of 1 μm to several hundreds of μm, and are surrounded by a thin film.
It is used in fields such as pesticides, fragrances, feeds, and foods. Microcapsules are generally encapsulated by forming a film on the outside of a substance with a certain property, and at the same time, encapsulating that property, and if a treatment such as destroying the film is performed when necessary. The substance can be released. Microcapsules can be manufactured by coacervation method, interfacial polymerization method, in situ
Methods, spray drying methods, in-liquid curing methods and the like are well known. On the other hand, some microcapsules using microorganisms that have been proposed so far are completely different manufacturing methods from these, and since the cell walls of microorganisms are used as membrane materials,
It is obtained by ingesting the substance to be encapsulated into the already prepared membrane material.

Specific examples of the method for producing microcapsules using microorganisms include the following. U.S. Pat. No. 4,001,480 discloses a method of encapsulating a fat-soluble substance by contacting it with a fungal having a proliferative function having a natural fat content of about 40 to 60% by weight. In Japanese Patent Laid-Open No. 58-107189, an aqueous slurry of growing microorganisms (yeast having a proliferative function) having a lipid content of 10% by weight or more recovered from a medium and an organic substance for increasing lipid content containing a dye (for example, having 4 carbon atoms). A method of contacting the above-mentioned aliphatic alcohols, esters, aromatic hydrocarbons, and other such organic solvents that are difficult or immiscible with water) in a two-layer system is disclosed. In addition, JP-A-61-8
No. 8871 discloses a liquid in which a hydrophobic substance (dye precursor) is dissolved or finely dispersed in a solvent and a fungus having a lipid content of less than 10% by weight (for example, dry yeast (dead yeast) which is highly thermally affected). A method of mixing in a two layer system is disclosed.
Further, in JP-A-63-88033, in yeast cells,
A method of allowing a monohydric alcohol having 1 to 3 carbon atoms to coexist in the process of microencapsulation (encapsulation) is disclosed in order to encapsulate a larger amount of hydrophobic liquid (for example, corn oil, alkylated naphthalene, etc.). .

However, these microencapsulation methods are basically aimed at encapsulation of dyes, fragrances, etc. In JP-A-5-95791, yeast cells are entrapped with lipids (oils and fats). Therefore, even if the above method is applied, the amount of lipids (oils and fats) encapsulated is extremely small at 5% by weight or less. Therefore, a method of encapsulating the lipids (oils and fats) after saponification or lipase treatment is proposed. . However, in this method, although a complicated operation such as saponification treatment or lipase treatment is performed, the decomposition products of fats and oils (that is, fatty acids) are only included in the end, and the fats and oils are essentially the molecules that should be present. It is not contained as it is. In addition, in JP-A-5-95791, a lipid (oil or fat) itself is emulsified in water to be encapsulated, or an ethanol / water (weight ratio 1: 1) system is encapsulated. It is described that the inclusion amount is at most 3% by weight.

The present inventors have made various studies on a method of incorporating lipids such as fish oil and vegetable oil as they are without using saponification treatment or lipase treatment, using yeast, with reference to conventionally proposed methods. However, JP-A-63-
It was found that it is extremely difficult to encapsulate a large amount as in Example 3 of 88033, and it is also extremely difficult to encapsulate a lipid as described in JP-A-5-95791. Thus, when the substances are included in yeast, not all the substances can be included by one method. In other words, the encapsulating method differs depending on the substance to be encapsulated, and the encapsulating cannot be efficiently performed unless an appropriate method is selected. As described above, the method for producing microcapsules using microorganisms is
Although various proposals have been made, a simple method for encapsulating a large amount of lipid has not been known so far. In addition, yeast containing phospholipids is not known.

[0006]

An object of the present invention is to develop yeast containing phospholipids. Preferably, in the encapsulation process of phospholipids, it is to develop a yeast containing a large amount of phospholipids without loss of useful substances produced in yeast cells to the outside of the cells.

[0007]

Means for Solving the Problems The inventors of the present invention have made extensive studies as to a method of encapsulating phospholipids using yeast, and as a result, saponification or lipase treatment of phospholipids to be encapsulated in yeast cells has been carried out. It was found that the desired yeast containing phospholipid can be rapidly obtained by contacting with yeast after forming a solution, suspension or emulsion containing phospholipid without treatment. Furthermore, when a considerable amount of hydrophilic organic liquid or hydrophobic organic liquid is used to prepare a solution or suspension containing phospholipids, some of the useful substances in the yeast cells are extracted and eluted in the organic liquid. Or, there are drawbacks such as relatively small inclusion amount of phospholipids, on the other hand, by contacting the emulsion of phospholipids and yeast in water without using the organic liquid at all or almost, It has been found that the drawbacks of can be overcome. That is, the present invention relates to a yeast containing an externally added phospholipid, a method for producing the yeast, and a feed using the yeast. The first of the production method, by contacting the yeast with an alcohol solution containing a phospholipid, the second, by dispersing the yeast in a mixed solvent system of a hydrophobic liquid in which the phospholipid is dissolved and water, further, The third is a method for producing a yeast containing a phospholipid, which is obtained by contacting yeast with a phospholipid emulsion in the presence of water.

The present invention will be described in detail below. The term “encapsulated” in the present invention means that yeast is used as a microcapsule film and the externally added substance is included in the cells. The yeast that can be used in the present invention is a general term for microorganisms that are taxonomically classified as yeast. Specifically, for example, Saccharomyces cerevisiae, Saccharomyces rouxii, Saccharomyces carlsberge
nsis), Candida tropic
alis), Candida lipoly
tica), Candida flaver
i), Candida curvata, Candida utills, Endomyces vernalis, Lipomyces lipofer, Lipomyces lipofer
Starkei (Lipomyces starkeyi), Trichosporon pullulans, Trichosporon
Yeast fungi such as, but not limited to, Trichosporon cutaneum, Phaffia rhodozyma, and Rodotorula glutinis can be used. Commercially available so-called baker's yeast, brewer's yeast,
It is also convenient to use yeast for feed. It can be used when the lipid content in yeast is high or low.

The preferred state of these yeasts used in the present invention depends on the method of encapsulation, but basically, they may be in a raw state, a dried state, or a dead state having no proliferative ability. In addition, heat treatment (for example,
Aging treatment at 15 to 200 ° C. (treatment of supplying oxygen with the supply of nutrients stopped after completion of culture), heat treatment]
You can also do Furthermore, by decomposing the cell wall,
It is also possible to use a yeast having enhanced permeability to the inside of the bacterial cell. Examples of the cell wall decomposition treatment method include treatment methods that have been known for a long time, for example, acid treatment at 20 to 100 ° C. using 0.1 to 6 N hydrochloric acid, sulfuric acid, or the like, sodium hydroxide at pH 8 to 14 or water. Use an aqueous solution of potassium oxide, etc. 2
Examples thereof include alkali treatment at 0 to 100 ° C. and enzyme treatment using an enzyme capable of dissolving cell walls such as Bacillus circulans. Regarding these various processing methods,
For example, JP-A-4-4033 and JP-A-4-63127.
Japanese Patent Laid-Open No. 4-117245, Japanese Patent Laid-Open No. 5-13801
No. 0. Yeast that can be used in the present invention,
It may be a mixture of these yeasts, or a mixture of these yeasts and mechanically crushed yeasts.

Examples of the phospholipid which can be used in the present invention include phosphatidylcholine and phosphatidylethanolamine derived from tissues of animal yolk, egg yolk, soybeans, etc.
Phosphatidylinositol, phosphatidylserine,
Natural or synthetic phospholipids such as sphingomyelin, etc., or egg yolk lecithin which is a mixture thereof, soybean lecithin, hydrogenated products thereof, or disteroylphosphatidylcholine obtained by semisynthesis can be used. These may be used alone or in combination. In addition to phospholipids, other lipid components (for example, fatty acids, alcohols, waxes, other complex lipids, cholesterol, vitamins, degradation products obtained by saponification or lipase treatment of phospholipids, other monoglycerides, diglycerides, triglycerides, etc.) ) Can also be used together.

First, the first manufacturing method will be described. In this production method, a yeast having a phospholipid encapsulated therein is obtained by contacting yeast with an alcohol solution containing a phospholipid. In this alcohol solution, there is no separated aqueous layer. Alcohols that can be used include monohydric alcohols having 1 to 4 carbon atoms, preferably monohydric alcohols having 1 to 3 carbon atoms, and most preferably ethanol. These alcohols may be used alone or in a mixture. The alcohol concentration in the system is generally preferably about 50% by weight or more. These alcohols have a relatively low boiling point, and are advantageous in that they can be easily removed by drying if they remain in the product. The above solution contains monohydric alcohols having 5 or more carbon atoms, other alcohols such as glycerin, water, halogenated hydrocarbons such as chloroform and methylene chloride, aliphatic hydrocarbons such as hexane and methylcyclohexane, acetone and methylisobutyl. It may contain ketones such as ketones, ethers such as diethyl ether, methyl tert-butyl ether and isopropyl ether, esters such as ethyl acetate, aromatic hydrocarbons such as toluene, and oils and fats to the extent that they have no influence. Further, a surfactant and an emulsifier can be added as needed. Although there is no limitation on the state of yeast that can be used, yeast that has been subjected to cell wall degradation treatment by the method described above is preferred.

The amount of phospholipid used is the amount to be encapsulated,
There is no particular limitation as it depends on the amount of solvent used and other conditions. Generally, about 5 to 1 relative to yeast (dry weight)
00% by weight is sufficient, but if necessary 100
You may use it by weight% or more. The cell concentration (dry weight) in the yeast suspension is not particularly limited, but is about 5 to 20% by weight.
It is a degree. When the cell concentration is low, production efficiency is poor, and
If the cell concentration is too high, stirring becomes difficult. PH when included
May be adjusted as necessary, but generally about 3 to 9 may be performed. Usually, it may be carried out at the pH of the culture medium or at a neutral pH. The temperature is not particularly limited and is, for example, in the range of about 10 to 70 ° C, usually 10 to 50 ° C.
It is preferably carried out to some extent. If necessary, the inclusion may be promoted by heating or pressurizing. The time depends on operating conditions such as temperature, pressure and stirring, but is about 30 minutes to 5 hours.

Next, the second manufacturing method will be described. In this production method, yeast is dispersed in a mixed solvent of a hydrophobic liquid in which phospholipid is dissolved and water to obtain yeast containing phospholipid. Examples of hydrophobic liquids that can be used include phospholipids such as aliphatic hydrocarbons such as hexane, petroleum ether and toluene, halogenated hydrocarbons such as chloroform and methylene chloride, ethers such as diethyl ether and dibutyl ether, and the like. A low to high boiling point solvent may be used. The above-mentioned hydrophobic liquid may contain other hydrophobic liquid, or acetone or C1-4.
Although it may contain a water-soluble liquid such as alcohol, the conditions under which the yeast is dissolved cannot be used. From the viewpoint of toxicity to the living body, if you want to minimize the residual solvent in the product,
It is preferable to use a solvent that can be easily removed by drying (for example, a boiling point of about 100 ° C. or lower). Further, a surfactant and an emulsifier can be added as needed. Incidentally, JP-A-63
No. 88033 discloses a method of increasing the amount of inclusion by allowing a monohydric alcohol having 1 to 3 carbon atoms to coexist, but in the second production method of the present invention, as shown later in Reference Examples, The inclusion amount of phospholipids does not increase even when these alcohols coexist. Although there is no limitation on the state of yeast that can be used, yeast in which the cell wall is decomposed by the above method is more preferable. The amount of phospholipid used depends on the amount to be encapsulated, the amount of solvent used and other conditions, and is not particularly limited. Generally, about 5 to yeast (dry weight)
~ 100% by weight is sufficient, but if necessary 1
You may use 100 weight% or more.

The ratio of water to the hydrophobic liquid and the yeast cell concentration (dry weight) are not particularly limited and may be appropriately selected depending on the production efficiency, the degree of stirring and the like. The pH at the time of encapsulation may be adjusted as necessary, but generally about 3 to 9 may be used. Usually, it may be carried out at the pH of the culture medium or at a neutral pH. The temperature is not particularly limited and is, for example, about 10 to 70.
The temperature is in the range of 0 ° C, and is usually preferably 10 to 50 ° C. If necessary, the inclusion may be promoted by heating or pressurizing. Since the liquid-liquid two-layer system is mixed, it is necessary to stir the mixture so that both layers are appropriately mixed. Time is temperature,
It takes about 30 minutes to 5 hours, depending on operating conditions such as pressure and stirring.

Next, the third manufacturing method will be described. The third production method is to obtain yeast containing phospholipid by contacting the yeast and phospholipid emulsion in the presence of water. As a method for preparing a phospholipid emulsion, a method using a surfactant or an emulsifier can be used. The type of surfactant or emulsifier is not particularly limited, for example, glycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, Hydroxypropyl cellulose, ethyl methyl cellulose,
Carboxymethyl cellulose and the like can be mentioned. In addition, an emulsion may be prepared using starch, gelatin, collagen, gum arabic, guar gum, alginate, a high molecular weight emulsifier having a molecular weight of 1000 or more, and stable emulsion is obtained. You just have to choose what you can achieve. By dissolving or dispersing the phospholipid in a small amount of other hydrophobic liquid such as lipid, the ease of solubilization and emulsification can be increased and the inclusion can be facilitated. These may be used alone or as a mixture of two or more. The amount of the above substance to phospholipid is
It cannot be specified because it depends on the types of the above substances, conditions such as stirring and temperature. For example, generally, it can be carried out at about 10% by weight or less with respect to the amount of phospholipid used, but the optimum amount for inclusion can be easily set.

The emulsification of the above-mentioned phospholipid can be carried out, as is generally known, by vigorously stirring and mixing the above-mentioned substance and an aqueous solution containing the phospholipid. For the stirring and mixing, a method suitable for emulsification can be selected, and for example, known methods such as a method of strongly stirring with a turbine blade, a method of using ultrasonic waves, a method of applying pressure, etc. are preferably used.

Further, as another method for preparing an emulsion of phospholipid, a vortexing method such as dissolving phospholipid in an organic solvent such as chloroform and sparging with nitrogen gas or distilling under reduced pressure or phospholipid having 1 carbon atoms is used. In addition to the method of dispersion-mixing in an aqueous solution containing alcohol of 4 to 4 or a mixture thereof, various methods generally used as a method for producing liposomes can be used (for example, JP-A-61-44808). Etc.). You may add surfactant and an emulsifier as needed. Further, by dissolving or dispersing the phospholipid in a small amount of another hydrophobic liquid such as lipid, solubilization and emulsification can be facilitated and the inclusion can be facilitated. The above-mentioned method for obtaining an emulsion of phospholipid is one example, and any other emulsion obtained by any method can be used.

The liquid containing the emulsion or liposomes thus prepared, or the solvent-free emulsion or liposomes can be included by mixing with yeast cells in the presence of water. Of course, an in situ method in which the emulsion preparation and the encapsulation are performed at the same time, and a method of alternately repeating the method can also be used. There are no particular restrictions on the yeast conditions that can be used. The amount of phospholipid to be used is not particularly limited because it depends on the amount and conditions of inclusion. Generally, about 5 to 100% by weight with respect to the yeast (dry weight) is sufficient, but if necessary, 100% by weight or more may be used. The yeast concentration is not particularly limited, but is about 5
It is 20% by weight. When the cell concentration is low, the production efficiency is low,
If the cell concentration is too high, stirring becomes difficult. The pH at the time of encapsulation may be adjusted as necessary, but generally about 3
It is sufficient to carry out from about 9 to 9. Usually, it may be carried out at the pH of the culture medium or at a neutral pH. The temperature is not particularly limited and is, for example, in the range of about 10 to 70 ° C., usually 10
It is preferably carried out at about 50 ° C. If necessary, the inclusion may be promoted by heating or pressurizing. Time is temperature,
It takes about 30 minutes to 5 hours depending on conditions such as pressure and stirring.

In the first and second aspects of the present invention, since a considerable amount of a hydrophilic organic liquid or a hydrophobic organic liquid is used to prepare a solution or suspension containing phospholipids, a useful substance in yeast cells is used. However, according to the third production method of the present invention, it is possible to use no or almost any organic liquid. Instead, the emulsion of phospholipid and yeast are brought into contact with each other in water, so that a part of the useful substances in the yeast cells can be prevented from being extracted / eluted into the organic liquid. In addition, the inclusion amount of phospholipid can be remarkably increased.
Furthermore, when phospholipids are used as liposomes, an extremely stable emulsified state of phospholipids can be obtained without the use of special emulsifiers or surfactants. It has great commercial advantages as it avoids the attachment of phospholipids to the shaft.

In the above first, second, and third methods of the present invention, phospholipids are the main components, and other lipid components such as fatty acids, alcohols, waxes, other complex lipids,
Cholesterol, vitamins, phospholipid saponification or lipase-decomposed products, other monoglycerides,
A broadly defined lipid component such as diglyceride or triglyceride can also be included. As a general lipid component used with phospholipids, widely used in animals, plants and microorganisms, ether mainly composed of glyceride, chloroform, a fraction soluble in a solvent having a relatively low polarity such as petroleum ether, for example, Land animal fats and oils such as lard and beef tallow, corn oil, vegetable fats and oils such as soybean oil and rapeseed oil, marine fats and oils such as cod oil, shishamo oil and sardine oil, Nocardia (No
cardia) genus bacteria, Candida lipolytica (Can
Microbial fats and oils produced by yeast such as dida lipolytica) can be used. Of course, components other than the lipid component may be included. As antioxidants and bioactive substances such as ethoxyquin, tocopherol and vitamin C, p
-Antifungal agents such as hydroxybenzoic acid and propionic acids,
Carotin, astaxanthin, carotenoids such as vitamin A and vitamin active substances, glutathione, ergosterol, ubidecarenone, etc., dyes, medicines,
A compound effective for pesticides, insecticides, fragrances, feeds, foods, etc. can be included to some extent. Needless to say again, in the practice of the present invention, if necessary, various treatments of phospholipids, various treatments of yeast and inclusion treatment may be performed separately, or may be performed simultaneously, and the most preferred method is selected. do it.

When the yeast used in the present invention is a dry yeast obtained by spray drying or the like, it is preferable that the yeast is soaked in water for a certain period of time, but there is a tendency that the amount of inclusion is generally small. However, in the practice of the present invention, it is considered that there is not much advantage in using dry yeast because water, alcohol, or other solvent is used, or since it is often finally dried after further encapsulation. However, dry yeast can be used if desired.

The yeast of the present invention thus obtained can be used as it is, but the solvent and unencapsulated phospholipids are washed with a solvent such as hexane, centrifuged, and distilled off under reduced pressure. You may remove and use it by a general method. Furthermore, these can be used as wet bacterial cells by a general method using a centrifuge, a filter, or the like,
Further, it can also be used as a dried bacterial cell or a dried granulated product obtained by a general method using a spray dryer, a drum dryer, a freeze dryer, a fluid dryer, or the like. Of course, during drying and / or granulation, for example, starch, gelatin, gum arabic, guar gum, dextrin, pectin, pullulan, casein, casein compounds, dried egg white, methylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropylmethylcellulose, It can be coated using a generally known coating medium or binder such as ethyl methyl cellulose, carboxymethyl cellulose, alginate, shellac, glucose, sucrose, fructose, lactose, maltose, starch sugar, etc., or together with other components including these. It can also be granulated. Further, for example, as described in JP-A-62-294079,
A film of a condensate such as an amino resin initial condensate (for example, a melamine-formalin initial condensate) may be formed and used. Of course, from the yeast of the present invention, ketones such as acetone and isobutyl ketone, aliphatic hydrocarbons such as hexane and petroleum ether, halogenated hydrocarbons such as methylene chloride, esters such as ethyl acetate, ethanol, n-propanol and isopropanol, etc. It is also possible to extract and elute the contents using the alcohol, the edible oil, etc., or a mixture thereof, and use the extracted contents.

The thus-obtained phospholipid-encapsulating yeast of the present invention, when compared with non-encapsulating yeast, is subjected to pressure such as being strongly pressed with a finger, and the encapsulated phospholipid and other lipid components. At the same time, the yeast contents are likely to bleed. Utilizing this property, the yeast of the present invention can be used in the fields of dyes, medicines, agricultural chemicals, perfumes, foods and the like. Further, it can be used as a mixed feed as it is or by mixing with a commonly used feed mixture. In particular, salmon, trout, effective improvement of carotenoids such as astaxanthin in yeasts such as Phaffia rhodozyma, which has been recognized as an effect of improving color tone and nutritional supplementation of red sea bream and crustaceans, and other effects in yeast When used to improve the absorbability of the ingredients, they are used as they are, or generally, the formulations of commonly used cultured seafood blended feeds such as fish meal, meat-and-bone meal, krill meal, soybean meal, corn gluten meal. , Feed oil, torula yeast, flour, starch, guar gum, dextrin, CM
It can be mixed with phospholipids such as C and lecithin, minerals, vitamins such as vitamin C and vitamin E, and molded into pellets or mash to prepare a compounded feed. As pellets, ordinary pellets (cold pellets,
In addition to hard pellets, EP (expanded pellets) can be used without particular limitation. In this case, the addition amount of the yeast containing the phospholipid of the present invention is generally effective at 10 to 150 ppm as astaxanthin, and may be appropriately adjusted depending on the purpose of improving the color tone. Traditionally,
Various cell wall treatment methods have been studied in order to effectively utilize carotenoids such as astaxanthin in astaxanthin-producing yeasts such as Phaffia rhodozyma to effectively improve coloration, but containing astaxanthin-producing yeast containing the phospholipid of the present invention The thus-produced feed for cultured seafood can be expected to have a higher effect of improving the color frying than the yeast obtained by the conventionally proposed treatment method.

[0024]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following description, all the weights of yeasts are weights in a dry state, and the contents of lipid components in yeast cells are all weight% on a dry matter basis. The phospholipids used in the following examples are soybean lecithin (soybean oil 25-
45% by weight, carbohydrates and sterols 3-7% by weight, phosphatidylcholine 15-25% by weight, phosphatidylethanolamine 10-20% by weight, phosphatidylinositol 15-25% by weight, phosphatidic acid 3-7.
% By weight, but the phospholipids that can be used in the practice of the present invention are not limited to this soy lecithin.

(Measurement Method of Encapsulation Amount) Yeast in the yeast suspension after the encapsulation treatment is allowed to settle, and the liquid layer containing the unencapsulated phospholipid is removed. The part of the yeast that has settled is added to water to obtain suspension water having a cell concentration of about 7% by weight. The suspension was washed with about the same amount of hexane as the hexane layer until no coloration was observed (about 5 times or more), the phospholipids adhering to the yeast were dissolved and removed, and the washed yeast was washed. (A lipid such as a phospholipid easily attaches to the yeast surface, and thus a lipid that does not elute even after washing with hexane is a truly encapsulated lipid). Yeast in this aqueous layer is allowed to settle to obtain a part of the settled yeast, which is concentrated under reduced pressure and further dried under vacuum. The dried product thus obtained is pulverized to obtain a powder (A). On the other hand, as a control sample, the yeast in which the phospholipid is not included is also vacuum dried to obtain the powder (B). After the powder is obtained, the amount of inclusion is measured by the following procedure.

(Measurement Method 1) About 1 g of the powders (A) and (B) is used to measure the water content with an infrared moisture meter. Approximately 4 g of powders (A) and (B) were soaked in 16 g of water, and then chloroform / methanol (2: 1)
The organic layer obtained by extraction with 400 ml, centrifugation and washing is concentrated under reduced pressure at 40 ° C. or lower and vacuum dried, and then the weight of the extract is measured. From the above, the amount of the extract of the powder (A) or (B) on the basis of 1 g of the dry matter is calculated. The value calculated according to the following formula is the inclusion amount. Encapsulation amount (%) = {[amount of extract (g) on the basis of 1 g of dry body of powder (A) -amount of extract (g) on the basis of 1 g of dry body of powder (B)] / [1 (g) -Amount of extract (g) based on 1 g of dry powder of powder (A)]} × 100

(Measurement method 2) About 1 g of the powders (A) and (B) is measured for moisture content with an infrared moisture meter. About 0.1 g of powder (A) and (B) 0.4 ml each
After soaking well with water, the mixture is extracted with 10 ml of chloroform / methanol (2: 1) and centrifuged to obtain an organic layer. The organic layer is analyzed by HPLC to measure the amount of phosphatidylcholine (PC amount). From the above, powder 1 (A), dry body 1 of (B)
Measure the amount of PC on a g basis. The value calculated according to the following formula is the inclusion amount. Inclusion amount (%) = {[PC on the basis of 1 g dry material of powder (A)]
Amount (g) -PC amount (g) of dry powder of powder (B) based on 1 g] / [PC content in 1 g of lecithin used (g) -based on dry product of powder (A) based on 1 g) PC amount (g)]} × 100

(Measurement Method 3) About 1 g of the powders (A) and (B) is measured for moisture content with an infrared moisture meter. About 20 mg of powders (A) and (B) are added to 1 ml of dimethylsulfoxide, and dissolved in an ultrasonic cleaner at 40 ° C. for 30 minutes. Next, 5 ml of acetone is added and mixed, and then the mixture is made up to 20 ml with acetone. The organic layer is obtained by centrifugation. The organic layer was analyzed by HPLC, and the amount of astaxanthin (A
X amount) is measured. Maximum 4% by weight of total AX in hexane cleaning solution
Since the following is extracted and removed, the AX amount in the hexane washing solution is also measured by HPLC analysis so as not to excessively evaluate the inclusion amount, and this is used for correction. The value calculated according to the following formula is the inclusion amount. Encapsulation amount (%) = {[AX on the basis of 1 g dry body of powder (B)]
Amount (g) / AX amount (g) on the basis of 1 g dry material of powder (A)
+ A in the hexane washing liquid when 1 g of dry powder (A) was prepared
X amount (g)]-1} × 100 When preparing the powder (A), a large amount of lipid-adhered components such as phospholipids are removed by washing with hexane, but the powder (B) has almost no lipid-adhered components. Since the dry weight remains almost unchanged after washing with hexane, washing with hexane at the time of preparing the powder (B) is not performed.

Example 1 2.5 g of lecithin was added to n-propanol / water (volume ratio 1:
2) Dispersed completely in 5 ml. Then, after adding 15 ml of water, the mixture was stirred at room temperature for about 1 hour until the fluidity of the liquid was restored. Separately, 50 g of suspension water containing 5 g of baker's yeast (live cells of Saccharomyces cerevisiae, lipid content 9% by weight) was prepared. This yeast suspension water was heat-treated at 50 ° C. for 2 hours, cooled to room temperature, and the above lecithin-containing solution was added. The mixture was stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 1, the amount of lecithin contained in yeast was 19% by weight. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 25% by weight.

Examples 2 and 3 5 g of the baker's yeast used in Example 1 was treated with acid in 45 ml of sulfuric acid having the normality shown in Table 1 at 70 ° C. for 100 minutes.
This solution was neutralized with an aqueous sodium hydroxide solution, centrifuged to collect the cells, and washed with a sufficient amount of water. 50 g of suspension water containing the obtained wet cells was prepared. The lecithin-containing solution obtained in the same manner as in Example 1 was added to this yeast suspension water, and the mixture was stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was as shown in Table 1.

[0031]

[Table 1]

Example 4 Baker's yeast (live cells of Saccharomyces cerevisiae,
Instead of 5 g of lipid content 9% by weight, Phaffia rhodozyma yeast (viable cells aged at 20 ° C. for 8 hours after culturing,
5 g of a lipid content (13% by weight) was used and encapsulated in the same manner as in Example 1. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 33% by weight.

Example 5 5 g of Phaffia rhodozyma yeast used in Example 4
50 g of suspension water containing (live cells) was prepared. The lecithin-containing solution obtained in the same manner as in Example 1 was added to this yeast suspension water, and the mixture was stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 5% by weight.

Examples 6, 7 and 8 Using 5 g of the Phaffia rhodozyma yeast used in Example 4, 60 ° C. in 45 ml of sulfuric acid having the normality shown in Table 2.
Acid treatment for 100 minutes. This solution was neutralized with an aqueous sodium hydroxide solution, centrifuged to collect the cells, and washed with a sufficient amount of water. 50 g of suspension water containing the obtained wet cells was prepared. The lecithin-containing solution obtained in the same manner as in Example 1 was added to this yeast suspension water, and the mixture was stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 3, the amount of lecithin contained in yeast was as shown in Table 2.

[0035]

[Table 2]

Example 9 Instead of 5 g of Phaffia rhodozyma yeast (live cells aged at 20 ° C. for 8 hours after cultivation, lipid content 13% by weight), Phaffia rhodozyma yeast (live cells after cultivation, lipid content (12% by weight) was included in the same manner as in Example 7 except that 5 g was used. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 21% by weight. Also,
As a result of analysis using measurement method 3, the amount of lecithin contained in yeast was 19% by weight.

Examples 10, 11, 12 5 g of Phaffia rhodozyma yeast used in Example 4
To the temperature and pH shown in Table 3
Alkali treatment in 5 ml for about 2 hours. After neutralizing this solution with hydrochloric acid, the cells were collected by centrifugation and washed with a sufficient amount of water. 50 g of suspension water containing the obtained wet cells was prepared.
The lecithin-containing solution obtained in the same manner as in Example 1 was added to this yeast suspension water, and the mixture was stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was as shown in Table 3.

[0038]

[Table 3]

Example 13 By the same method as in Example 7, yeast suspension water having a bacterial cell concentration of 10% by weight of Phaffia rhodozyma yeast was obtained. This suspension water is dried with a spray dryer (hot air inlet 180 ° C),
Dried cells (lipid content 18% by weight, water content 6% by weight) were obtained. 50 g of suspension water containing 5 g of the dried cells was prepared. The lecithin-containing solution obtained in the same manner as in Example 1 was added to this suspension water, and the mixture was stirred at room temperature for 3 hours for inclusion. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 13% by weight.

Example 14 About 5000 ml of 500 g of Phaffia rhodozyma yeast
Of water and adjusted to pH 4.5 with acetic acid. 25 g of fancerase (trade name of Yakult Pharmaceutical Co., Ltd.) was added, and the mixture was stirred at room temperature for about 3 hours, but the enzyme treatment seemed to be incomplete under these conditions. The treated liquid was neutralized with an aqueous sodium hydroxide solution and dried with a spray dryer (hot air inlet 180 ° C.) to obtain dried bacterial cells (water content 7%). 50 g of suspension water containing 5 g of the dried cells was prepared.
The lecithin-containing solution obtained in the same manner as in Example 1 was added to this suspension water, and the mixture was stirred at room temperature for 3 hours for inclusion. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 5% by weight.

Example 15 A lecithin-containing solution obtained in the same manner as in Example 1 was freeze-dried to remove the solvent, and a cotton-like product was obtained. This was added to 50 g of suspension water containing 5 g of Phaffia rhodozyma yeast obtained by the same method as in Example 7, and stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 19% by weight.

Example 16 2.5 g of lecithin was added to and mixed with 4.4 ml of n-propanol. This was added to 50 g of suspension water containing 5 g of Phaffia rhodozyma yeast obtained by the same method as in Example 7, and vigorously stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 14% by weight.

Example 17 0.1 g of sucrose fatty acid ester was added to 20 g of water.
Further, 2.5 g of lecithin was added and dispersed, and the mixture was emulsified with a homogenizer. This emulsion was suspended in water containing 5 g of Phaffia rhodozyma yeast obtained in the same manner as in Example 7 and suspended in water 50.
g, and the mixture was stirred at room temperature for 3 hours for inclusion. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 17% by weight. Unlike Examples 1 to 16, some adhesion of lecithin whose emulsion was broken was observed on the stirring shaft.

Example 18 Example 17 except that 0.1 g of hydroxypropyl cellulose was used instead of 0.1 g of sucrose fatty acid ester.
It was included as well. As a result of analysis using measurement method 2,
The content of lecithin in yeast was 19% by weight. Unlike Examples 1 to 16, some adhesion of lecithin whose emulsion was broken was observed on the stirring shaft.

Example 19 50 g of suspension water containing 5 g of Phaffia rhodozyma yeast (viable cells) was prepared. After heat treatment at 50 ° C for 2 hours,
Cooled to room temperature. To this, a lecithin emulsion obtained in the same manner as in Example 17 was added, and the mixture was stirred at room temperature for 3 hours for inclusion. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 6% by weight. By stirring at high speed, it was possible to suppress the adhesion of the emulsified broken lecithin to the stirring shaft.

Examples 20, 21, 22, 23, 24 Acid-treated wet cells of Phaffia rhodozyma yeast were obtained in the same manner as in Example 7. 2.5g of this yeast 5g
Was added to 50 ml of the alcohol shown in Table 4 containing lecithin. The amount of water introduced from the wet cells is about 15
Was ~ 25 g. The mixture was stirred and mixed at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was as shown in Table 4. In addition, contents such as astaxanthin in Phaffia rhodozyma yeast were eluted in the alcohol solution.

[0047]

[Table 4]

Example 25 Heat-treated wet cells of Phaffia rhodozyma yeast were obtained in the same manner as in Example 4. Using 5g of this yeast,
It was encapsulated in the same manner as in Example 21. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 5% by weight.

Example 26 Acid-treated wet cells of Phaffia rhodozyma yeast were obtained in the same manner as in Example 7. 25 g of suspended water containing 5 g of this yeast (containing 20 g of water) was prepared. Also, 2.5g
Of lecithin was added to and mixed with 20 g of ethanol. Both liquids were mixed and mixed with stirring at room temperature for 3 hours to be included.
As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 10% by weight.

Examples 27, 28, 29, 30 2.5 g of lecithin was dissolved in 20 g of the solvent shown in Table 5. This was added to 50 g of suspension water containing 5 g of Phaffia rhodozyma yeast obtained by the same method as in Example 7, and stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was as shown in Table 5. In Examples 27 and 28, the elution of the contents such as astaxanthin in the Fafire Rhodotima yeast was observed in a relatively large amount, but in Examples 29 and 30, the elution amount was considerably small.

[0051]

[Table 5]

Example 31 5 g of Fafire rhodozyma yeast used in Example 9
In 45 ml of 2.0 N sulfuric acid at 60 ° C.
Acid treatment for 00 minutes. The solution was neutralized with an aqueous solution of sodium hydroxide, centrifuged to collect the liquid, and washed with a sufficient amount of water. 50 g of suspension water containing the obtained wet cells was prepared.
On the other hand, a mixture of 2.5 g of lecithin and 0.25 g of Dunaliella carotene (Kyowa Hakko, trade name, containing 4% by weight of extracted carotene and 96% by weight of vegetable oil) was added to 5 ml of n-propanol / water (volume ratio 1: 2). Disperse completely, then water 15
After adding ml, the mixture was stirred at room temperature for about 1 hour until the fluidity of the liquid was restored. This lecithin-containing solution was added to the above-mentioned yeast suspension water, and stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the inclusion amount was 27% by weight, and as a result of analysis using measurement method 3, the inclusion amount was 23%.
It was found to be 4% by weight, and it was found that the content of carotene in yeast was improved four times.

Example 32 The same procedure as in Example 31 was carried out except that 0.25 g of 1-menthol was used instead of 0.25 g of Dunaliella carotene. As a result of analysis using measurement method 2, the amount of lecithin included was 22% by weight, and when the wet bacterial cells after washing with hexane were crushed with a finger, they smelled as 1-menthol.

Example 33 The procedure of Example 31 was repeated, except that 0.25 g of ethoxyquine was used instead of 0.25 g of Dunaliella carotene. The content of lecithin in the yeast was 26% by weight as a result of analysis of the entrapped components in yeast using the measuring method 2. Also, H
As a result of PLC analysis, ethoxyquin was also detected.

Example 34 The procedure of Example 31 was repeated except that 0.10 g of glutathione was used instead of 0.25 g of Dunaliella carotene. As a result of HPLC analysis of the entrapped components in yeast, glutathione was detected.

Example 35 An encapsulation was carried out in the same manner as in Example 31 except that 0.10 g of ergosterol was used instead of 0.25 g of Dunaliella carotene. As a result of HPLC analysis of the inclusion components in yeast, ergosterol was detected.

Example 36 Encapsulation was carried out in the same manner as in Example 31 except that 0.10 g of ubidecarenone was used in place of 0.25 g of Dunaliella carotene. As a result of HPLC analysis of the inclusion components in yeast, ubidecarenone was detected.

Example 37 Similar to Example 7, Faffire rhodozyma yeast 20
An encapsulation treatment solution was prepared using 0 g. After adding 40 g of glucose to the solution to dissolve it, the solution was dried with a spray dryer (hot air inlet 180 ° C.) to obtain dried cells.

Example 38 As in Example 4, Faffire rhodozyma yeast 20
An encapsulation treatment solution was prepared using 0 g. This solution was washed with the same volume of hexane until the hexane layer became almost colorless. The remaining hexane was removed under reduced pressure, 40 g of glucose was added and dissolved, and then dried with a spray dryer (hot air inlet 180 ° C.) to obtain dry cells.

Example 39 As in Example 11, Faffire rhodozyma yeast 2
An encapsulation treatment liquid was prepared using 00 g. 40g in this liquid
After adding glucose and 100 g of dextrin,
It was dried with a spray dryer (hot air inlet 180 ° C.) to obtain dried cells.

Example 40 Four large test tubes containing 5 ml of a medium were inoculated with Fafire rhodozyma yeast and cultured at 20 ° C. for 48 hours. The contents were transferred to four 500 ml Sakaguchi flasks containing 50 ml of medium and cultured at 20 ° C. for 48 hours. 25 contents
It was transferred to a 5000 ml mini jar containing 00 ml of medium and cultured at 20 ° C. for 48 hours. The contents were transferred to a 50-liter culture tank containing 25 liters of medium, and main culture was carried out. In the main culture, the pH was controlled between 4.8 and 5.5, and the dissolved enzyme concentration was maintained between 30 and 80% of saturation. Glucose as a carbon source was added at the start of 500 g, and after the glucose was consumed, the glucose was supplemented. The initial supplementation was slow, then gradually faster so that no glucose remained. (Medium) KH ₂ PO ₄ : 0.7 (% by weight, the same applies hereinafter), (NH ₄ )
2HPO ₄ : 1.3%, MgSO ₄ 7H ₂ O: 1530ppm, ZnSO
_{4 · 7H 2 O: 170ppm,} MnSO 4 · 4H 2 O: 17ppm, NaC
l: 170ppm, CaCl ・ 2H ₂ O: 300ppm, FeSO ₄・ 7H ₂
O: 30 ppm, CuSO ₄ .5H ₂ O: 1.5 ppm, yeast extract: 0.3%, foaming agent: 100 ppm, pH adjusting: ammonia water The addition of glucose was stopped after about 100 hours of culture. 1
After 0 hours, take about one-fifth of the culture solution, add concentrated sulfuric acid to a sulfuric acid concentration of 2.5 N, oxidize and decompose at 100 ° C for 100 minutes, and then neutralize with aqueous sodium hydroxide solution. did. Then, the cells were collected by centrifugation and washed with a sufficient amount of water. Using this microbial cell, add water to obtain a microbial cell concentration of 10
% Bacterial cell suspension (containing 250 g of dry cells) 2
500 g was obtained. 125 g of soybean lecithin was completely dispersed in 250 ml of n-propanol / water (volume ratio 1: 2). Then, after adding 750 ml of water, the mixture was stirred at room temperature for about 1 hour until the fluidity of the liquid was restored. The lecithin-containing solution was added to the cell suspension, and the mixture was stirred at 30 ° C. for 3 hours to be included. As a result of analysis using measurement method 3, the amount of lecithin included was 24% by weight. Next, after adding 50 g of glucose to the above-mentioned encapsulation treatment solution to dissolve it, spray-drying (inlet temperature: 170 ° C.) using a spray drier to obtain dried microbial cells of lecithin-encapsulating Fafire rhodozyma yeast. It was

Reference Examples 1, 2, 3 and 4 were encapsulated using the solvents shown in Table 6 in the same manner as in Examples 27, 28, 29 and 30 except that 4 g of n-propanol was added and coexisted. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was as shown in Table 6.
As is clear from the results in Table 6, the addition of n-propanol did not increase the amount of inclusion.

[0063]

[Table 6]

Reference Example 5 Lecithin (2.5 g) was dissolved in hexane (20 g).
-7 g of propanol was added and coexisted. The suspension was added to 70 g of suspension water containing 5 g of Fafire rhodozyma yeast obtained by the same method as in Example 7, and the mixture was stirred at room temperature for 3 hours to be included. As a result of analysis using measurement method 2, the amount of lecithin contained in yeast was 2% by weight. As is clear from this result, the addition of n-propanol did not increase the amount of inclusion.

Comparative Example 1 Approximately ⅕ of the culture broth obtained in Example 40 was taken, treated with acid and neutralized in the same manner as in Example 40, and the cells were collected to obtain a cell concentration of 10% by weight. A body suspension was prepared. This suspension is spray-dried using a spray dryer (inlet temperature 170 ° C)
Then, a dried microbial cell of Fafire rhodozyma yeast containing no lecithin was obtained.

Comparative Example 2 The procedure of Example 17 was repeated except that 2.5 g of corn oil was used instead of 2.5 g of lecithin. As a result of analysis using measurement method 1, the amount of corn oil contained in yeast was 3% by weight. In addition, as a result of analysis using measurement method 3,
The amount of corn oil contained in yeast was 4% by weight.

Comparative Example 3 The same procedure as in Comparative Example 1 was carried out except that 4 g of n-propanol was added at the time of emulsification. As a result of analysis using measurement method 3, the amount of corn oil contained in yeast was 0% by weight.

Application Example 1 Using the dried bacterial cells of each of the Fafire rhodozyma yeasts produced in Example 40 and Comparative Example 1, a feed pellet having the composition shown in Table 7 was prepared, and a rainbow trout fried test was conducted. It was

[0069]

[Table 7]

Test fish: A rainbow trout having an average weight of 400 g and a uniform weight. Breeding method Facility: Concrete water tank of 3.6 m × 0.9 m × 0.9 m is set in one water tank per test area. Water: Water temperature of 15 ° C. Groundwater is used, water injection rate is 15 liters / minute, and there is aeration. Number of fish to be tested Tail fish: 40 fish per aquarium Feeding amount: Feeding is divided into 3 times based on approximately 2% of the fish weight per day Evaluation item: Every evaluation day General breeding performance, sensory evaluation of meat color and color fan (Hoffman La.
Table 8 shows the flesh color evaluation results (average value) of the evaluation test using a color card manufactured by Roche.

[0071]

[Table 8]

◯: Good color deepening and good color tone. X: The color is deep, but the color is light. In addition, general breeding results did not show any abnormalities in each ward. As is clear from the results shown in Table 8, the product of the present invention has a good frying effect.

[0073]

INDUSTRIAL APPLICABILITY According to the present invention, a highly useful yeast containing phospholipid can be obtained by a simple method and is useful for feed, food, medicine, agricultural chemicals and the like.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12N 1/06 8828-4B 1/16 J 8828-4B // A23J 7/00

Claims (14)

[Claims] 1. A yeast containing an externally added phospholipid. 2. The yeast according to claim 1, wherein at least one selected from the group consisting of bioactive substances, antioxidants, dyes, and ingredients effective in foods is included together with the externally added phospholipid. 3. The yeast according to claim 1 or 2, wherein the yeast has been subjected to a cell wall decomposition treatment. 4. The phospholipid is lecithin, claims 1-3.
The yeast described. 5. The yeast according to claim 1, wherein the yeast is an astaxanthin-producing yeast. 6. The yeast according to claim 5, wherein the astaxanthin-producing yeast is Phaffia rhodozyma. 7. A method for producing a yeast containing an externally added phospholipid, which comprises contacting the yeast with a solution containing a phospholipid-containing monohydric alcohol having 1 to 4 carbon atoms. 8. The alcohol concentration in the alcohol solution is 5
The method for producing a yeast according to claim 7, wherein the amount is 0% by weight or more. 9. A method for producing yeast containing an externally added phospholipid, wherein the yeast is dispersed and mixed in a two-layer liquid of a hydrophobic liquid in which the phospholipid is dissolved and water. 10. A method for producing a yeast containing an externally added phospholipid, which comprises contacting an emulsion of phospholipid and yeast in the presence of water. 11. A phospholipid emulsion comprising a surfactant and / or
Or an emulsion prepared by using an emulsifier.
Production method described in 0. 12. The production method according to claim 10, wherein the phospholipid emulsion is dispersed and mixed in an alcohol having 1 to 4 carbon atoms or an aqueous solution of the alcohol and water. 13. A feed comprising the phospholipid-encapsulating yeast according to any one of claims 1 to 6. 14. The feed according to claim 1, which is a feed for cultured seafood.
The feed described in 3.