JP2023091644A - Yeast powder flavor for food and drink, and method for preparing the same - Google Patents

Abstract

Kind Code: A1 A powdery yeast flavor with improved inclusion amount of a hydrophobic flavor and a method for preparing the same are provided.
A yeast cell wall fraction having a median diameter D50 of 1 μm to 10 μm in a volume-based particle size distribution measured for an aqueous dispersion using a laser diffraction/scattering particle size distribution analyzer is included in a hydrophobic perfume. use. As the hydrophobic perfume to be clathrated, a perfume having an octanol/water partition coefficient (logP) of 1.5 to 4.8 is used.
[Selection figure] None

Description

TECHNICAL FIELD The present invention relates to a yeast powder flavor for food and drink, which is obtained by enclosing a hydrophobic flavor in yeast cells, and a method for preparing the same.

Conventionally, as a method of controlling the release timing and rate of a desired component to increase the sustainability of the component, the component is enclosed in a container (microcapsule) having a diameter of about several μm to several hundred μm made of a natural or synthetic polymer. , so-called microencapsulation methods are known. Yeast microcapsules using yeast cell walls as capsule shells are also widely known as microcapsules using this technology.

As a method for producing yeast microcapsules, a method of including a desired exogenous component such as a fragrance as it is without removing the content component (cell content) of the yeast body (for example, Patent Publication 1 etc.), and yeast There is a method of including a desired exogenous component in the cells after removing the contents of cells in the body (for example, Patent Publications 2 to 6, etc.). The latter method is useful in that the more the cell content in the yeast cells is removed, the more the exogenous component can be included, and as a result, the more the action and effect of the exogenous component can be enjoyed. be.

Furthermore, as a method for enjoying more of the effects of the exogenous component enclosed in the yeast cells, a part or the entire surface of the yeast microcapsule prepared by the latter method (exogenous component inclusion yeast) , sugars, high-intensity sweeteners, proteins, and polyhydric alcohols are attached (Patent Documents 7 and 8). By doing so, it is possible to prevent deterioration of the exogenous component in the yeast cells, and to control the speed, strength, or duration of its release outside the yeast cells.

JP-A-61-88871 JP-A-4-4033 JP-A-4-63127 JP-A-4-117245 JP-A-5-15770 JP-A-8-243378 International Publication 2003/041509 Japanese Patent Application Laid-Open No. 2004-24042 JP-A-8-243378 Japanese Patent Publication No. 7-32871 Japanese Patent Publication No. 8-29246

The present invention relates to the above-described yeast microencapsulation technology, and an object of the present invention is to enclose a larger amount of hydrophobic flavoring agents in yeast cells. Specifically, an object of the present invention is to provide a powdery flavoring agent for food and drink in which a large amount of hydrophobic flavoring agent is included in yeast cells, and a method for preparing the same.

In order to solve the above problems, the present inventors used a plurality of commercially available yeasts sold by various companies to perform a treatment to remove cellular contents in the cells and a treatment to include a hydrophobic fragrance. When we measured the amount of hydrophobic fragrance actually enclosed in the cells (inclusion rate), we confirmed that there was variation in the inclusion rate. It was found that the particle size distribution of the bacteria is the cause. Therefore, with the aim of improving the encapsulation rate of hydrophobic flavorings, further studies were carried out. By adjusting the median diameter D50 of the formula (volume basis) to be in the range of 1 μm to 10 μm, regardless of whether the yeast sold by each company is different, the clathration rate of the hydrophobic flavor is generally improved. I have found that the goal is achievable.

The present invention has been completed based on such findings, and has the following embodiments.
(I) Yeast cell preparation used for flavor inclusion
The yeast cell preparation used for flavor inclusion in the present invention is obtained by partially destroying the cell membrane of the yeast cell to extract the cellular contents (organelles, intracellular matrix, etc.) contained in the yeast cell. is the yeast cell wall fraction released outside the cell. The yeast cell wall fraction is usually used in a state of being suspended in water for encapsulating the flavor. The term "yeast cell wall fraction" also includes, but is not limited to, yeast cell wall fractions in such aqueous suspensions.
In the present invention, the term “inclusion” refers to a state in which the target flavoring component permeates the inside of the yeast cell wall (that is, the space filled with the intracellular matrix in the yeast before treatment).

The yeast cell wall fraction (yeast cell preparation) has the following characteristics.
(I-1) Yeast cells used for encapsulating flavorings, wherein the median diameter D50 in the volume-based particle size distribution measured for the water suspension is 1 μm to 10 μm using a laser diffraction/scattering particle size distribution analyzer. Mural minute.
(I-2) The yeast cell wall fraction according to (I-1), wherein the 10% diameter (D10) and 90% diameter (D90) in the particle size distribution are in the range of 1 μm to 10 μm.
(I-3) The yeast cell wall fraction according to (I-1) or (I-2), which has an average particle diameter of 1 μm to 10 μm and/or a mode diameter of 1 μm to 10 μm in the particle size distribution.

(II) Method for preparing yeast cell wall fraction used for flavor inclusion (II-1) Subjecting yeast cells to treatment including elution of cell contents in the cells and fractionation,
Fractions with a median diameter D50 of 1 μm to 10 μm in the volume-based particle size distribution measured for the aqueous suspension are fractionated using a laser diffraction/scattering particle size distribution analyzer to obtain yeast cell wall fractions. having a step of
The method for preparing the yeast cell wall fraction according to any one of (I-1) to (I-3).

(III) The yeast cell wall fraction described in any of (I-1) to (I-3) for food and drink powdered flavoring is used as a yeast cell from which cell contents have been removed, and hydrophobic It can be used as a powdered flavor for food and drink by enclosing a natural flavor. The powder flavoring agent for food and drink includes the following aspects.
(III-1) A powdered flavoring agent for food and drink, in which a hydrophobic flavoring agent is included in yeast cells from which cell contents have been removed,
The yeast cells have a median diameter D50 of 1 μm to 10 μm in the volume-based particle size distribution measured for the water suspension using a laser diffraction/scattering particle size distribution analyzer. Product powder perfume.
(III-2) The powdery flavorant for food and drink according to (III-1), wherein the hydrophobic flavorant has an octanol/water partition coefficient (logP) of 1.5 to 4.8.
(III-3) The hydrophobic perfume is at least one perfume component selected from the group consisting of acids having 6 to 12 carbon atoms, aldehydes, esters and alcohols, (III-1) or (III-2) Powder flavor for food and drink described in .
(III-4) The food and drink according to any one of (III-1) to (III-3), wherein the hydrophobic flavor is at least one selected from the group consisting of menthol, menthol analogues, and orange flavor. Product powder perfume.
(III-5) The powdered flavoring for food and drink according to any one of (III-1) to (III-4), which is a flavoring for chewing gum.

(IV) Method for preparing yeast powder flavor for food and drink (IV-1) The yeast cell wall fraction described in any of (I-1) to (I-3) has an octanol/water partition coefficient (logP) of 1. (III-1) to (III-5), which has a step of preparing a yeast powder flavor in which the hydrophobic flavor is included in the yeast cell wall fraction by contacting the hydrophobic flavor of .5 to 4.8. A method for producing a powdered flavoring agent for food or drink according to any one of the above.
(IV-2) A step of attaching at least one selected from the group consisting of sugars, high intensity sweeteners, proteins and polyhydric alcohols to the surface of the hydrophobic flavor inclusion yeast (IV-1 ).

(V) Method for improving inclusion property of hydrophobic flavor in yeast cell preparation used for flavor inclusion (V-1) A method for improving inclusion property of hydrophobic flavor in yeast cell wall fraction, comprising: The cell wall fraction is adjusted (granulated) so that the median diameter D50 in the volume-based particle size distribution measured for the aqueous suspension using a laser diffraction/scattering particle size distribution analyzer is 1 μm to 10 μm. The above method, characterized in that:

By using the yeast cell wall fraction prepared by the method of the present invention for encapsulation of a hydrophobic flavor for food and drink, it is possible to prepare a yeast powder flavor with a higher flavor inclusion rate than conventionally. In addition, by using the yeast cell wall fraction prepared by the method of the present invention, it is possible to improve the variation in the yeast cells used as the starting material and to uniformly enclose the hydrophobic flavor. Since the yeast powder flavor has a high inclusion rate (amount) of the hydrophobic flavor, it exhibits a high effect as a flavor in foods and drinks, especially products that are chewed in the oral cavity such as chewing gum. be able to.
Furthermore, by attaching at least one selected from the group consisting of sugars, high-intensity sweeteners, proteins and polyhydric alcohols to the surface of the yeast powder flavor, the known effect (release of hydrophobic flavor control of timing and speed) can be exhibited more effectively.

The results of measuring the particle size distribution of the supernatant fraction and the precipitate fraction of each yeast raw material used in Experimental Example using a laser diffraction particle size distribution analyzer Microtrac are shown (Experimental Example 1). (a) yeast A, (b) yeast B, (c) yeast C, (d) yeast D, (e) yeast E. In each figure, the thin line indicates the particle size distribution of the supernatant fraction, and the thick line indicates the particle size distribution of the precipitate fraction. In Experimental Example 3, the particle size distributions of the supernatant fraction and the precipitate fraction of the yeast cell wall fractions (derived from yeast A and C) prepared using the method before improvement and improved method 2 were measured using a laser diffraction particle size distribution analyzer micrometer. Shows the results measured using the track. (a) derived from yeast A (improved method 2), (b) derived from yeast A (unimproved method), (c) derived from yeast C (improved method 2), (d) derived from yeast C (unimproved method). In each figure, the thin line indicates the particle size distribution of the supernatant fraction, and the thick line indicates the particle size distribution of the precipitate fraction. 4 shows the results of measuring the particle size distribution of the yeast supernatant fraction using a laser diffraction particle size distribution analyzer Microtrac in Experimental Example 5. FIG. (a) Yeast cell wall fraction when mixed with a highly clathrate flavoring agent for a certain period of time, (b) Yeast cell wall surface when stirring with a lowly clathrate agent for a certain period of time using an inverted microscope (600x magnification). (Experimental Example 6).

(I) Yeast Cell Wall Fraction Used for Flavor Encapsulation and Preparation Method Thereof The yeast cell wall fraction used for flavor inclusion targeted by the present invention is used as a container for containing a desired hydrophobic flavor inside. from which the cell contents have been removed to accommodate the hydrophobic flavorant. The yeast cell wall fraction used for flavor inclusion is usually used in the form of an aqueous suspension with a solid content of about 10%.
Since the yeast cell wall fraction is highly stable, if it is not used for inclusion of a hydrophobic fragrance for a long period of time (for example, if 4 parts of ethanol is added to 96 parts of the suspension, about 12 months), Once, it can be stored in a dry powder state by a method such as spray drying. The dry powdery yeast cell walls can be dispersed again in water before being used for encapsulating the hydrophobic flavoring agent.

Individual yeast cell wall particles contained in the yeast cell wall fraction of the present invention have a volume-based particle size distribution when the aqueous suspension of the yeast cell wall is measured using a laser diffraction/scattering particle size distribution analyzer. It is characterized by a median diameter D50 of 1 μm to 10 μm.

The yeast cell wall fraction of the present invention preferably has the following particle size distribution and average particle size. Each of these means a volume-based particle size distribution calculated by measuring the particle size distribution of an aqueous suspension of the yeast cell wall using a laser diffraction/scattering particle size distribution analyzer. The solid content in the water suspension to be measured is adjusted to 1 to 5% by mass so that the TR value (transmittance) when subjected to a particle size distribution meter is in the range of 0.75 to 0.95. bottom. Within this range, no difference occurs in the detected particle size distribution. In addition, the measurement method includes a wet method (a method of measuring by dispersing powder in an insoluble solvent) and a dry method (a method of directly measuring powder), but the values specified in the present invention are both It is a value measured by a wet method using a flow system using water as a solvent.

[Median diameter D50]
It is usually 1 μm to 10 μm, preferably 1 to 9 μm, more preferably 2 to 8 μm.
[10% diameter D10]
It is usually 0.1 μm to 5 μm, preferably 0.2 to 4.9 μm, more preferably 0.3 to 4.8 μm.
[90% diameter D90]
It is usually 4 μm to 10 μm, preferably 4.1 to 9.9 μm, more preferably 4.2 to 9.8 μm.
[Particle size and frequency (%)]
Proportion of particles of 1 μm or less: 70% or less, preferably 50% or less, more preferably 0-30%.
Proportion of particles of 10 μm or more: 50% or less, preferably 40% or less, more preferably 0-30%.
[Volume average diameter]
It is usually 1 μm to 10 μm, preferably 1 to 9 μm, more preferably 2 to 8 μm.
[Mode diameter]
It is usually 1 μm to 10 μm, preferably 1 to 9 μm, more preferably 2 to 8 μm.

The yeast used for the preparation of the yeast cell wall fraction (hereinafter also referred to as "yeast raw material" or simply "yeast") may be suitable for ingestion or administration by the human body. Yeasts such as baker's yeast, wine yeast, and torula yeast that have been conventionally used for producing oral compositions such as food and drink can be arbitrarily used. Specifically, yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, Saccharomyces rouxii, and Saccharomyces carlsbergensis; Candida utilis, Candy Yeast belonging to the genus Candida, such as Candida tropicalis, Candida lipolytica, and Candida flaveri; Torulaspora delbrueckii, Torulaspora fermentati fermentati), and Torula yeast such as Torulaspora rosei. These yeasts may be used alone, or two or more of them may be used in any combination. Any of these yeast raw materials can be used as raw materials for producing the yeast cell wall fraction of the present invention, regardless of whether they are viable or dead, or wet or dry.

The yeast cell wall fraction of the present invention is not limited, but the yeast raw material is subjected to a treatment for eluting cell contents in the cells (cell contents elution treatment) and a fractionation treatment, and the median diameter in the particle size distribution described above is It can be prepared by fractionating a fraction having a D50 of 1 μm to 10 μm.

"Cell content elution treatment" is a process that elutes endogenous intracellular components, including amino acid components, peptide and protein components (including enzymes), carbohydrate components, nucleic acid components, and lipid components, out of the cells. be. The method is not particularly limited as long as the yeast cell wall to be prepared has chemical and physical properties that are acceptable as a microcapsule shell and this can be achieved, and it is carried out by arbitrarily combining known methods. can do. For example, known methods include heating treatment; elution accelerator addition method; enzymatic treatment method using enzymes such as intracellular component eluting enzymes; physical treatment method such as pH treatment; A combination of the above processes can be mentioned (for example, see Patent Documents 7 and 8). A combination of enzyme treatment and heat treatment is preferred, and a method of combining these treatments with pH treatment is more preferred.

For example, although the heating treatment is not limited, it is carried out by heating a liquid obtained by suspending the raw yeast material in water to usually 30 to 100° C., preferably 30 to 60° C., and stirring for several minutes to several hours. be able to. At this time, an elution accelerator can be used in combination for efficient elution of cell contents from the cells. Examples of such elution accelerators include lower alcohols such as ethanol; polar organic solvents such as ethyl acetate and acetone; and the like.

Enzymatic treatment methods include a method utilizing autolytic enzymes possessed by yeast (Babayan, T.L. and Bezrukov, M.G., 1 Acta Biotechnol.0, 5, 129-136 (1985)), protease alone, nuclease, β-glucanase. , esterase or lipase in combination with a protease to treat yeast. Specifically, it can be carried out by incubating an aqueous dispersion of yeast containing an autolyzing enzyme or an aqueous dispersion of yeast to which the enzyme has been added at a temperature of 30 to 60° C. for about 1 to 48 hours.

For the pH treatment, the target yeast raw material is suspended in an acidic aqueous solution (pH 2 or less) such as hydrochloric acid, phosphoric acid, sulfuric acid, lactic acid, citric acid, acetic acid, or ascorbic acid, and heated (50 ~ 100 ° C.); and an alkaline treatment method in which the target yeast raw material is stirred in an alkaline aqueous solution, preferably an aqueous solution having a pH of 9 to 13, more preferably pH 10 to 12, over several minutes to several hours. be The temperature of the alkaline aqueous solution is not particularly limited, and usually in the range of 20 to 100°C can be used. Inorganic salts such as sodium hydroxide, calcium hydroxide, potassium hydroxide and sodium silicate; or organic nitrogen compounds such as ammonia, monoethanoldiamine, ethylenediamine and diethylenetriamine aqueous solutions can be used to prepare the alkaline aqueous solution. The pH treatment is preferably an acid treatment under heating.

Examples of the alcohol treatment include a method of adding a monohydric alcohol to the target yeast raw material.

Both the pH treatment and the alcohol treatment are steps adopted as pretreatments of yeast cells for inclusion of as many exogenous components as possible inside the yeast cells (Patent Documents 9 to 11). After enzymatically treating the raw material, the supernatant can be removed, and the resulting bacterial cell residue can be washed, if necessary, and then subjected to pH treatment and/or alcohol treatment. Acid treatment is preferred.

The yeast cell wall preparations prepared by these cell content elution treatments can then be fractionated to fractionate fractions having a median diameter D50 of 1 μm to 10 μm in the particle size distribution described above.
Such a fractionation treatment may be a treatment for obtaining a yeast cell wall fraction having a median diameter D50 in the water suspension within the above range from the yeast cell wall preparation prepared by the cell content elution treatment. . Preferably, among the yeast cell wall preparations, the median diameter D50 in the aqueous suspension is in the above range, the 10% diameter (D10) is in the range of 0.1 μm to 5 μm, and the 90% diameter (D90) is A method for obtaining fractions in the range of 4 μm to 10 μm. More preferably, the yeast cell wall preparation has a median diameter D50 in the water suspension in the above range, a mode diameter in the range of 1 μm to 10 μm, and/or a volume average diameter in the range of 1 μm to 10 μm. is a method of obtaining fractions at Preferred ranges and more preferred ranges of the median diameter D50, 10% diameter (D10), 90% diameter (D90), mode diameter, and volume average diameter are as described above.

The fractionation treatment is not particularly limited as long as the yeast cell wall obtained has chemical and physical properties that can be used as a microcapsule shell and the above can be achieved. Although not limited, for example, fractionation processing using centrifugation, sorting processing using a membrane filter or the like, field flow fractionation processing such as flow cytometry, fractionation processing using electrophoresis technology such as electrophoresis, etc. can be carried out singly or in combination. Centrifugation is preferred because of its simple operation. The conditions employed in each of these fractionation treatments can be appropriately set so that the prepared yeast cell wall satisfies the particle size distribution described above.

These fractionation treatments may be performed after the cell contents elution treatment described above, or may be performed before the cell contents elution treatment. Moreover, when two or more cell content elution treatments are performed in combination, the treatment can be performed between these treatments.

Although the method for preparing the yeast cell wall fraction is not limited, preferably, the yeast cells used as the yeast raw material are subjected to fractionation treatment, enzymatic treatment under heating conditions, acid treatment under heating conditions, A method of performing a summation treatment and a fractionation treatment to obtain a sedimentation residue as a yeast cell wall suspension can be mentioned. More preferably, the fractionation treatment is performed not only at the beginning and end, but also between the enzymatic treatment and the acid treatment, and between the acid treatment and the neutralization treatment, and the sedimentation residue obtained by the fractionation treatment is subjected to the next step. It is a method of providing Here, the fractionation treatment preferably includes centrifugation treatment.

Centrifugation conditions are not limited as long as the above purpose can be achieved. A method of centrifuging at 4000 to 8000 rpm for 5 to 20 minutes, preferably at 400 to 5000 rpm for 5 to 15 minutes can be exemplified.

The thus-prepared yeast cell wall fraction can uniformly accommodate a large amount of the hydrophobic flavoring agents described later in the cells regardless of the yeast used as a raw material (yeast raw material). It is useful as a cell wall fraction.
The form of the yeast cell wall fraction may be an aggregate of the dried powder of the yeast cell wall in a dispersed state (dry powder aggregate), and the yeast cell wall does not damage the yeast cells such as water. It may be aggregated in a solvent-dispersed state (solvent dispersion).

(II) Yeast powder flavor and its preparation method The yeast powder flavor of the present invention includes a hydrophobic flavor having an octanol/water partition coefficient (logP) of 1.5 to 4.8 in the yeast cell wall fraction described above. An aqueous suspension of hydrophobic flavored yeast is spray-dried with optional excipients.

"Octanol/water partition coefficient" (hereinafter also simply referred to as "Pow") is the chemical It means the concentration ratio of a substance, and is used as a physicochemical index that expresses the hydrophobicity (easily soluble in lipids) of a chemical substance. It is generally described in logarithmic value (logP).

As described above, the hydrophobic flavoring agent to be included inside the yeast cell wall fraction preferably has a logarithmic value (logP) of Pow in the range of 1.5 to 4.8. More preferably, logP = 1.7 to 4.77, still more preferably logP = 1.78 to 4.57, and particularly preferably logP = 1.8 to 4.0. If the logP value is less than 1.5, it is speculated that due to the low hydrophobicity, the flavor will dissolve in the suspension before inclusion, making it unsuitable for inclusion in the yeast cell wall fraction. In addition, when the logP value is greater than 4.8, it is assumed that due to the high hydrophobicity, it is not compatible with the suspension and is not suitable for inclusion in the yeast cell wall fraction.
The form of the hydrophobic perfume that can be used in the present invention is not particularly limited, but it may be crystalline at room temperature.

Specific examples of perfume ingredients satisfying the Pow logarithmic value (LogP) include hexanoic acid (n-caproic acid), octanoic acid (caprylic acid), decanoic acid (capric acid), and lauric acid (lauric acid ) and other acids having 6 to 12 carbon atoms (specifically carboxylic acids, more specifically linear saturated fatty acids); aldehydes having 6 to 10 carbon atoms such as hexanal, octanal, and decanal; butyl acetate, hexyl acetate , and esters with 6 to 10 carbon atoms such as octyl acetate; alcohols with 6 to 12 carbon atoms such as hexanal, octanol, decanol and dodecanol; . Acids, aldehydes, esters, alcohols and monoterpenes having 6 to 10 carbon atoms are preferred, and acids, aldehydes, esters, alcohols having 8 to 10 carbon atoms and monoterpenes having 10 carbon atoms are more preferred. The perfume targeted by the present invention may be composed of one of these perfume components alone, or may be composed of a combination of two or more of them.

Menthol, menthol analogues, and orange flavor are preferred. Analogs of menthol include, for example, l-menthol, dl-menthol, 3-l-menthoxypropane-1,2-diol, isopulegol, neoisopulegol, isoisopulegol, neoisoisopulegol, neomenthol, iso menthol, neoisomenthol, citronellol, linalool, mint oil, peppermint oil, menthyl lactate, isopulegol, menthyl succinate and the like. On the other hand, orange fragrance is a fragrance composition containing D-limonene as the main fragrance ingredient, but it also contains α-pinene, β-pinene, sabinene, myrcene, linalool, γ-terpinene, etc. can also be used in the present invention.

The hydrophobic flavoring agent can be included in the yeast cell wall fraction by being brought into contact with, that is, mixed with, the yeast cell wall fraction. Specifically, a hydrophobic flavoring agent is added to an aqueous suspension of the yeast cell wall portion, the pH and temperature are adjusted as desired, and the mixture is stirred for a predetermined time to allow the hydrophobic flavoring agent to be included in the yeast cell wall. be able to.
When the hydrophobic fragrance that can be used in the present invention has a crystal shape at room temperature, the conditions for inclusion in the yeast cell wall fraction include a temperature and a pH that allow the crystals to slowly dissolve in a liquid state. Conditions and stirring conditions may be adopted. Specifically, the following conditions are mentioned.
Although the pH is not particularly limited, it can be appropriately selected from the range of usually pH 5-9, preferably pH 6-8. The temperature is not particularly limited, but can be appropriately selected from the range of usually 40 to 80°C, preferably 50 to 70°C. Although the stirring is not limited, for example, by using various stirring devices such as a blender with stirring blades, an emulsifier, a disperser, or a homogenizer, the hydrophobic flavor is efficiently included in the yeast cell wall fraction. can be At this time, the stirring speed, stirring rotation speed, etc. are not particularly limited, but can be appropriately selected and adjusted from the range of usually 1,000 to 10,000 rpm, preferably 1,000 to 5,000 rpm.

When mixing the yeast cell wall fraction and the hydrophobic flavoring agent, a hardening agent, an antioxidant, a stabilizer, a dispersant, an emulsifier, and a pH adjuster are added to the system as long as the effects of the present invention are not hindered. , preservatives, anti-deterioration agents, or the like can also be added.

Then, if necessary, sugars and high-intensity sweeteners are added to part or all of the surface of the hydrophobic flavor-encapsulated yeast obtained by the above method (hereinafter referred to as "flavor-encapsulated yeast preparation"). , proteins and/or polyhydric alcohols can also be attached.

Sugars used here include monosaccharides such as arabinose, galactose, xylose, glucose, sorbose, fructose, rhamnose, ribose, isomerized liquid sugar, and N-acetyldarcosamine; isotrehalose, sucrose, trehalulose, trehalose, neotrehalose , palatinose, maltose, melibiose, lactulose, lactose, etc.; trisaccharides, such as maltotriose; tetrasaccharides, such as maltotetraose; triose, panose, etc.), oligo-N-acetyldarcosamine, galactosyl sucrose, galactosyl lactose, galactopyranosyl (β1-3) galactopyranosyl (β1-4) glucopyranose, galactopyranosyl (β1-3) glucopyranose, galactopyranosyl (β1-6) galactopyranosyl (β1-4) glucopyranose, galactopyranosyl (β1-6) glucopyranose, xylo-oligosaccharides (xylotriose, xylobiose, etc.), gentio-oligosaccharides ( gentiobiose, gentiotriose, gentiotetraose, etc.), stachyose, theandeoligo, nigerooligosaccharide (nigerose, etc.), palatinose oligosaccharide, palatinose syrup, fucose, fructo-oligosaccharide (kestose, nystose, etc.), fructofuranosylnistose, poly Dextrose, maltosyl β-cyclodextrin, maltooligosaccharides (maltotriose, tetraose, pentaose, hexaose, heptaose, etc.), raffinose, sugar-bound starch syrup (coupling sugar), soybean oligosaccharides, invert sugar, starch syrup, dextrin, gum arabic , xanthan gum, guar gum, carrageenan, curdlan, tamarind seed gum, dielan gum, starch, oligosaccharides and polysaccharides such as modified starch; isomaltitol, erythritol, xylitol, glycerol, sorbitol, palatinit, maltitol, maltotetri Examples include sugar alcohols such as tall, maltotriitol, mannitol, lactitol, reduced isomaltooligosaccharide, reduced xylo-oligosaccharide, reduced gentio-oligosaccharide, reduced maltose starch syrup, and reduced starch syrup. Preferred are disaccharides such as lactose and trehalose, and sugar alcohols such as mannitol. These saccharides can be used singly or in any combination of two or more.

High-intensity sweeteners include α-glucosyltransferase-treated stevia, aspartame, acesulfame potassium, alitame, licorice extract (glycyrrhizin), triammonium glycyrrhizinate, tripotassium glycyrrhizinate, trisodium glycyrrhizinate, diammonium glycyrrhizinate, Dipotassium glycyrrhizinate, disodium glycyrrhizinate, curculin, saccharin, sodium saccharin, cyclamate, sucralose, stevia extract, stevia powder, zuretin, thaumatin (thaumatin), Tenryocha extract, Neisseria veri extract, neotame, neohesperidin dihydrochalcone, Examples include fructosyltransferase-treated stevia, Brazilian licorice extract, miracle fruit extract, swingle extract, enzyme-treated licorice, and enzyme-decomposed licorice. These high-intensity sweeteners may be used singly or in any combination of two or more.

Proteins may include gelatin, gelatin hydrolysates, corn protein, casein, sodium caseinate, and collagen. These proteins can be used singly or in any combination of two or more.

Polyhydric alcohols include glycerin, propylene glycol, and their polymers polypropylene glycol and polyglycerin. These polyhydric alcohols may be used singly or in combination of two or more.

Further, these saccharides, high-intensity sweeteners, proteins and polyhydric alcohols can be used alone or in any combination of two or more. The method of attaching sugars, high-intensity sweeteners, proteins and polyhydric alcohols (hereinafter collectively referred to as "sugars, etc.") to the surface of the flavor clathrate yeast preparation described above is also not particularly limited. For example, the flavor clathrate yeast preparation prepared by the above-mentioned method is dried by a spray dryer or the like, and a solution or dispersion of sugars dissolved or dispersed in a solvent such as water is added to the preparation by a fluidized bed granulator or the like. A method of spraying by: Spraying an aqueous dispersion of a flavor clathrate yeast preparation toward a floor (fluid bed) spread with sugars, etc., and then spraying a dispersion of a flavor clathrate yeast preparation surface-covered with sugars, etc. A method of separating and drying the droplets; A method of adding sugars, etc. to the aqueous dispersion of the flavor-encapsulated yeast preparation and dissolving it uniformly, followed by freeze-drying; A method of drying and powder-mixing saccharides or the like with a high-speed stirring mixer or the like; and a method of adding saccharides or the like to an aqueous dispersion of a flavor clathrate yeast preparation, dissolving it uniformly, and then spraying the dispersion. A drying method and the like can be exemplified. Here, the ratio of the flavor-encapsulating yeast preparation to the saccharides, etc., is 1 to 90 parts by mass, preferably 1 to 90 parts by mass of the saccharides, etc. per 100 parts by mass of the hydrophobic flavor-encapsulating yeast preparation, in terms of the weight ratio of the dry solids. A proportion of 10 to 50 parts by weight, more preferably 20 to 40 parts by weight can be mentioned.

The thus-prepared flavor-encapsulating yeast preparation can be used as a component of foods, pharmaceuticals, quasi-drugs, cosmetics, or feeds. Preferably, it can be used as a flavoring component for oral compositions (food and drink, oral pharmaceuticals or quasi-drugs, and feed). Preferably, it can be used as a powder flavor (microencapsulated flavor) for food and drink. Here, the oral composition includes both an edible composition that can be ingested into the body and an oral composition that is used in the oral cavity regardless of the presence or absence of ingestion.

The powdered flavoring agent of the present invention can be suitably used as a compounding component of a food or drink that is maintained in the oral cavity or taken while being chewed in the oral cavity. Specific examples of such foods and drinks include chewing gum, soft candies such as gummies and nougat, and confectionery and luxury goods (food compositions) that are ingested by long chewing in the oral cavity. In addition, the chewing gum in the present invention broadly means gum that is chewed and ingested in the oral cavity, and is a concept including bubble gum.

The food and drink of the present invention, especially chewing gum, contain conventionally known food and drink, especially chewing gum ingredients, and can be produced according to a conventionally known production method, except that it contains the yeast powder flavor of the present invention as a component.
In the case of chewing gum, the proportion of the powdered yeast flavor of the present invention contained in 100% by weight of the chewing gum finally obtained is not limited, but can be usually selected from the range of 0.05 to 10% by weight.

When used for chewing gum, a specific formulation using the yeast powder flavor of the present invention is, for example, as follows.
[Prescription]
Gum base 25 parts Sugar 62 parts Corn syrup 10 parts Glycerin 1 part
Yeast powder flavoring 2 parts
A total of 100 parts Gum base, sugar, corn syrup and glycerin are mixed, yeast powder flavor is added, and the mixture is uniformly kneaded with a mixer while being kept at 50°C. After cooling, it is pressed and formed with a roller to prepare a plate-shaped chewing gum.

(III) Method for Improving Inclusion Properties of Hydrophobic Flavors in Yeast Cell Wall Fractions The present invention improves the inclusion properties of hydrophobic flavoring agents in yeast cell wall fractions used for flavor inclusion, and improves the inclusion properties of hydrophobic flavoring yeast cell wall surfaces. To provide a method for improving the encapsulation rate of a hydrophobic perfume in a minute.
In this method, the median diameter D50 in the volume-based particle size distribution measured for the aqueous suspension of the yeast cell wall fraction used for inclusion of the hydrophobic flavoring agent using a laser diffraction/scattering particle size distribution analyzer is It can be carried out by adjusting the grain size to be 1 μm to 10 μm (granule regulating treatment). Such a granule sizing treatment can be performed by using the fractionation treatment described in (I) above.

Furthermore, it is preferable to use a yeast cell wall fraction having the following particle size distribution and average particle size as the yeast cell wall fraction used for encapsulating the fragrance.
[Median diameter D50]
It is usually 1 μm to 10 μm, preferably 1 to 9 μm, more preferably 2 to 8 μm.
[10% diameter D10]
It is usually 0.1 μm to 5 μm, preferably 0.2 to 4.9 μm, more preferably 0.3 to 4.8 μm.
[90% diameter D90]
It is usually 4 μm to 10 μm, preferably 4.1 to 9.9 μm, more preferably 4.2 to 9.8 μm.
[Particle size and frequency (%)]
Proportion of particles of 1 μm or less: 70% or less, preferably 50% or less, more preferably 0-30%.
Proportion of particles of 10 μm or more: 50% or less, preferably 40% or less, more preferably 0-30%.
[Volume average diameter]
It is usually 1 μm to 10 μm, preferably 1 to 9 μm, more preferably 2 to 8 μm.
[Mode diameter]
It is usually 1 μm to 10 μm, preferably 1 to 9 μm, more preferably 2 to 8 μm.

The method for preparing such a yeast cell wall fraction is as described in (I) above, and the description thereof can be incorporated herein. The hydrophobic flavoring agent to be included in the yeast cell wall fraction is as described in (II) above, and the description thereof can be incorporated herein. Pow is preferably logarithmic (logP) in the range of 1.5 to 4.8, more preferably logP = 1.7 to 4.8, more preferably logP = 1.78 to 4.57, particularly preferably logP = hydrophobic perfume in the range of 1.8 to 4.0.

According to the method of the present invention, it is possible to improve the encapsulation rate of the hydrophobic flavoring agent in the yeast cell wall fraction used for encapsulating the flavoring agent.

As used herein, the terms "include" and "contain" include the meanings of "consisting of" and "consisting essentially of".

Hereinafter, the present invention will be described using experimental examples in order to facilitate understanding of the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were performed at room temperature (25±5° C.) and atmospheric pressure conditions. Unless otherwise specified, "%" described below means "% by mass", and "part" means "parts by mass".

The yeast raw materials and measurement methods used in the experimental examples are as follows.
[Yeast raw material]
(1) Yeast A: Wine yeast powder (S. cereviciae) (Nobless, Lallemand Inc.)
(2) Yeast B: Wine yeast powder (S. cereviciae) (OptiWhite, Lallemand Inc.)
(3) Yeast C: Brewer's yeast (S. cereviciae) (dried brewer's yeast HB-P02, Asahi Group Foods Co., Ltd.)
(4) Yeast D: Brewer's yeast (S. cereviciae) (dried brewer's yeast B, Miwa Pharmaceutical Co., Ltd.)
(5) Yeast E: Torula yeast powder (Candida utilis) (Miwa Pharmaceutical Co., Ltd.)

[Measurement of particle size distribution of yeast]
100 g of slurry prepared by dispersing 5 g of the yeast to be measured in water was centrifuged at 4500 rpm for 10 minutes to separate the supernatant and precipitate. The supernatant fraction obtained by fractionation was suspended in water to prepare an aqueous suspension with a solid content of 1% after measuring the amount of dry solids. On the other hand, the precipitate fraction obtained by fractionation was suspended in water to prepare an aqueous suspension having a solid content of 5% after measuring the amount of dry solids. The particle size distribution (volume basis) of the aqueous suspension of each fraction was measured with a laser diffraction particle size distribution meter Microtrac MT-3000II (Microtrac Inc.) (solvent refractive index: 1.333, particle refractive index: 1.81).

[Measurement of flavor content of flavor clathrate yeast]
(1) Measurement method of fragrance content (total oil amount) (outline)
The target flavor-encapsulated yeast powder was suspended in water:ethanol (50:50) or water:acetone (50:50), sonicated, and the flavor content in the prepared solution was quantified by gas chromatography.
(2) Measurement method of fragrance content (surface oil amount) (outline)
After stirring and suspending the subject flavor-encapsulated yeast powder in n-hexane, the filtrate was filtered using filter paper, and the flavor content in the filtrate was quantified by gas chromatography.
(3) Gas chromatography conditions GC device 6890N: manufactured by Agilent Technologies Column: DB-WAX/length 30 m, inner diameter 0.25 mm (manufactured by Agilent Technologies)
Column temperature conditions: 50 to 220°C (5°C/min temperature increase) (menthol)
40 to 200°C (2°C/min temperature rise) (D-limonene)
Carrier gas: helium.
A calibration curve was prepared in advance using known concentrations of various fragrances to be measured (standard product) and 1-octanol (internal standard product), and quantified by the calibration method.

Experimental Example 1 Measurement of particle size distribution of various yeast raw materials Yeast A to E described above as yeast raw materials were fractionated into supernatant and precipitate by the method described above, and the particle size distribution of each fraction sample (water suspension) was measured with a laser diffraction particle size distribution meter. The particle size distribution of _each fraction of each yeast raw material is shown in FIG.

As a result, it was confirmed that there is a difference in particle size distribution depending on the type of yeast, and even with the same type of yeast, depending on the manufacturer. However, it was confirmed that particles with a median diameter (D50) in the range of 1 to 10 µm were present in all yeast precipitate fractions. In the precipitated fractions of yeasts A and E, particles with a median diameter (D50) in the range of 1 to 10 μm and particles in the range of 10 to 100 μm were confirmed. This was considered to be in the state of agglomeration of particles (single particles) of 1 to 10 µm.

Experimental Example 2 Preparation of menthol-encapsulated yeast (powder flavoring) and its evaluation Using the above-described yeasts A to E as yeast raw materials, menthol (logP: 3.38) is introduced into the yeast cells in place of the cell contents. A menthol clathrate yeast (powder flavor) was prepared by clathration, and the content of menthol incorporated into the cells (menthol clathration rate) was measured.

(1) Preparation of menthol clathrate yeast (1-1) Removal of cell contents in yeast cells 100 g of various yeast raw materials (yeast A to E) are dispersed in water, (Amano Enzyme Co., Ltd.) was added, and the mixture was shaken at 50°C for 20 hours to elute out the cell contents in the cells. The resulting slurry was centrifuged at 8000 rpm for 20 minutes and the supernatant was removed to obtain 350 g of yeast cells (20% dry solid content). To this, 1036 g of water and 14 g of concentrated hydrochloric acid were added, heated at 80° C. for 30 minutes (acid treatment), and then cooled. After that, the mixture was centrifuged at 8000 rpm for 20 minutes, and the supernatant was removed to obtain 500 g of acid-treated yeast cells (dry solid content: 10%). A 10% sodium hydroxide aqueous solution was added to this to adjust the pH to 7.0 and neutralize. After that, the mixture was centrifuged at 8000 rpm for 20 minutes to remove the supernatant, and the residue was obtained as yeast from which the intracellular contents had been removed.

(1-2) Inclusion of menthol in yeast cells The thus-prepared cell content-free yeast was dispersed in water, and 450 g of the aqueous dispersion (dry solid content: 10%) was heated to 60°C. , 25 g of menthol was added to this and stirred at 3000 rpm for 2 hours to allow the menthol to clathrate in the cells of the yeast. Next, 60 g of a 50% lactose-containing aqueous solution heated to 60° C. was added to 475 g of the yeast-containing aqueous solution and mixed. Next, these mixed solutions were spray-dried in a spray dryer at an inlet temperature of 150° C. and an outlet temperature of 90° C. to obtain 90 g of menthol-encapsulated yeast powder (hereinafter simply referred to as “menthol-encapsulated yeast”). . Theoretically, the total powder should be 100 g, which means that 10 g was not recovered during the spray drying process.

<Composition of menthol clathrate yeast>
Yeast cell wall: 45 parts by mass Menthol: 25 parts by mass
Excipient (lactose): 30 parts by mass
Whole: 100 parts by mass

(2) Determination of menthol content in cells (2-1) Determination of menthol contained in menthol-encapsulated yeast (total) 0.1 g of each menthol-encapsulated yeast prepared above was added to 25 mL of ultrapure water. After suspension, sonication was performed for 5 minutes. 12.5 mL of 99% ethanol aqueous solution was added to the solution, and ultrasonic treatment was performed for 5 minutes. Next, 0.5 mL of 10% 1-octanol solution (solvent: ethanol) was added to this as an internal standard, 12 mL of 99% ethanol solution was added to prepare a test solution, and the menthol content was quantified by gas chromatography (GC). This content corresponds to the menthol content (total content) contained in the entire menthol clathrate yeast. From this total content, the ratio to 100% by mass of the total menthol clathrate yeast was calculated, and this was defined as the total content (%).

(2-2) Determination of menthol adhering to the surface of menthol-encapsulated yeast 5 g of each menthol-encapsulated yeast prepared in (1-2) above was suspended in 25 mL of n-hexane and stirred with a stirrer for 30 seconds. , as a suspension. Then, filter the suspension with filter paper, add 0.5 mL of 10% 1-octanol solution to the obtained filtrate as an internal standard, add 24.5 mL of n-hexane to make a test solution, and measure menthol by GC. The content was quantified. This content corresponds to the menthol content (surface content) adhering to the surface of the menthol clathrate yeast. From this surface content, the ratio to 100% by mass of the total menthol clathrate yeast was calculated, and this was defined as the surface content (%).

(2-3) Results From the quantitative results described above, the inclusion rate of menthol contained in the cells of the menthol-encapsulated yeast was calculated according to the following formula.
[Number 1]
Inclusion rate (%) = [(whole content rate (%)) - (surface content rate (%))] / (whole content rate (%))

Table 2 shows the results obtained for each yeast.

As shown in Table 2, the inclusion rate of menthol varied depending on the yeast species used. As shown in Experimental Example 1, the yeast cells sold by each company have variations in the particle size distribution of the cells due to differences in the type or purification process, which affects the inclusion rate of menthol in the cells. It was considered possible that

Experimental Example 3 Preparation of improved menthol clathrate yeast and its evaluation In order to improve the inclusion rate of menthol in yeast cells, yeasts A and C were used as yeast raw materials, and experimental example 2 (1) (1-1) We improved the method of "removal of cell contents in yeast cells" adopted in .

(1) Improvement method (1-1) Improvement 1
150 g each of yeast A and C were dispersed in water, and 3,000 g of slurry was well suspended, centrifuged at 4,500 rpm for 10 minutes, and the supernatant was removed to obtain 2,000 g of yeast cells (5% dry solids). (Step 1). By this centrifugation, particles with a median diameter of 0.1-1 μm contained in yeasts A and C were removed.
Then, the method described in Experimental Example 2 (1) (1-1) (Step 2) and (1-2) (Step 3) was performed to obtain menthol-encapsulated yeast powder (“menthol-encapsulated yeast”). ) was obtained (Example 1: derived from yeast A, Example 2: derived from yeast C).

(1-2) Improvement 2
150 g each of yeast A and C were dispersed in water, and 3,000 g of slurry was well suspended, centrifuged at 4,500 rpm for 10 minutes, and the supernatant was removed to obtain 2,000 g of yeast cells (5% dry solids). (Step 1).
Next, 2 g of Protease M "Amano" SD (Amano Enzyme Co., Ltd.) was added to this, and the mixture was shaken at a temperature of 50°C for 20 hours to elute out the cell contents inside the cells. The resulting slurry was centrifuged at 4500 rpm for 10 minutes and the supernatant was removed to obtain 350 g of yeast cells (20% dry solids). To this, 1036 g of water and 14 g of concentrated hydrochloric acid were added, heated at 80° C. for 30 minutes (acid treatment), and then cooled. After that, the mixture was centrifuged at 4500 rpm for 10 minutes, and the supernatant was removed to obtain 500 g of acid-treated yeast cells (dry solid content: 10%). A 10% sodium hydroxide aqueous solution was added to this to adjust the pH to 7.0 and neutralize. After that, the mixture was centrifuged at 4500 rpm for 10 minutes, the supernatant was removed, and the residue was obtained as yeast from which the intracellular contents had been removed (step 2′).
Then, the method described in Experimental Example 2 (1) (1-2) was performed (step 3) to obtain 90 g of menthol-encapsulated yeast powder (abbreviated as "menthol-encapsulated yeast") (Example 3: derived from yeast A, Example 4: derived from yeast C).

(2) Results (2-1) Quantitation of the menthol content included in the cells of the menthol-encapsulated yeast prepared above (Examples 1 to 4), by the method described in Experimental Example 2 (2). The menthol content in the whole body and the cell surface was quantified, and the total menthol content (%), surface content (%), and inclusion rate (%) were determined.
The results are shown in Table 3 together with the results of Experimental Example 2 (Comparative Example 1: derived from yeast A, Comparative Example 2: derived from yeast C).

As shown in Table 3, by adding a centrifugation step (step 1) at 4500 rpm for 10 minutes to the "removal of cell contents in yeast cells" method of Experimental Example 2 (1) (1-1) , an increase in menthol inclusion rate was observed (Examples 1 and 3). Therefore, yeast raw materials having a median diameter (D ₅₀ ) in the range of 0.1 to 1 μm should be subjected to an additional step of removing particles in the range, for example, by centrifugation at 4500 rpm for 10 minutes. , it was confirmed that the amount of menthol inclusion increased by the subsequent menthol inclusion treatment. Furthermore, in addition to the above step 1, in the "removal of cell contents in yeast cells" method described in Experimental Example 2 (1) (1-1), two centrifugation conditions (8000 rpm × 20 minutes) , 4500 rpm×10 min (Step 2), a further increase in menthol inclusion rate was observed (Examples 2 and 4).

(2-2) Particle size distribution measurement of yeast cell wall fraction Regarding the menthol clathrate yeast obtained above (Example 3: derived from yeast A, Example 4: derived from yeast C), the preparation steps (steps 1 and 2') ) For the fraction (yeast cell wall fraction) obtained in (improved method 2), the supernatant and residue (precipitate) obtained in step 2′ were separated, and the aqueous suspension of each fraction was treated with a laser. The particle size distribution was measured with a diffraction particle size distribution meter. For comparison, the corresponding yeasts A and C were treated in the same manner as the supernatant and the yeast from which the fungal cell contents were removed by the method (unimproved method) described in Experimental Example 2 (1) (1-1). The residue (precipitate) was fractionated, and the particle size distribution of each sample was measured using a laser diffraction particle size distribution analyzer.

The particle size distribution of _each yeast cell wall fraction is shown in FIG.

Examples 3 and 4 showed a higher menthol inclusion rate than Comparative Examples 1 and 2 (Table 3). On the other hand, the menthol inclusion rates in Examples 3 and 4 were higher than those in Examples 1 and 2, respectively.
As shown in Table 4 and FIG. 2, the yeast cell wall fractions prepared by improved method 2 (used as raw materials for Examples 3 and 4) all had almost the same particle size distribution between the precipitate and the supernatant. Both supernatants were confirmed to have a single peak in the range of 1-10 μm. In contrast, for all yeast cell wall fractions prepared by the unimproved method, the particle size distributions of the pellet and the supernatant were different, and the particle size distribution of the supernatant was smaller than that of the pellet.
From the above results, the yeast powder prepared so that both the precipitate and the supernatant have a single peak in the range of 1 to 10 μm is used as a raw material, and menthol is included in this. It was confirmed that the ratio (amount of inclusion) increased.

Experimental Example 4 Influence of cell inclusion rate due to difference in particle size distribution of yeast cells According to the above experimental example, yeast powder with a median diameter (D ₅₀ ) in the range of 1 to 10 μm was used as a raw material. , it was confirmed that the inclusion rate of the fragrance in the cells (perfume inclusion rate) amount is improved. Here, in order to confirm this again, the inclusion rate of menthol was measured using yeast cells having a peak at 1 μm or less in the particle size distribution.

(1) Preparation of Menthol-encapsulated Yeast Powder 1,080 g of a slurry obtained by dispersing 300 g of yeast A in water was centrifuged at 4,500 rpm for 10 minutes to obtain 450 g of supernatant (dry solid content: 10%). Thereafter, 90 g of menthol-encapsulated yeast powder (menthol-encapsulated yeast) was obtained by the methods described in Experimental Example 2 (1) and (1-2) (Comparative Example 3).

(2) Quantitation of Content of Included Menthol With regard to the obtained menthol-encapsulated yeast (Comparative Example 3), the menthol content of the whole cells and surface thereof was quantified in the same manner as in Experimental Example 2 (2). The results are shown in Table 5 together with the results of Comparative Example 1.

(3) Particle size distribution measurement Part of 450 g of the supernatant obtained in the preparation method of (1) above was measured for particle size distribution using a laser diffraction particle size distribution meter. The results are shown in FIG. From this, the median diameter (D ₅₀ ) of the particle size distribution was 0.45 µm.

(4) Results As shown above, Comparative Example 3 was confirmed to have a lower menthol content than Comparative Example 1 (Table 5). From this, the yeast powder present in the median diameter (D ₅₀ ) of 1 to 10 μm in the particle size distribution has a high fragrance inclusion ability, whereas the yeast powder present in the median diameter (D 50 ) of 1 μm or less has a low fragrance inclusion ability. It was observed that it does not contribute to the improvement of the fragrance encapsulation ability, and rather, it has a negative effect on the fragrance encapsulation ability because it is mixed in the yeast powder present in the median diameter (D ₅₀ ) of 1 to 10 μm. .

Experimental Example 5 Inclusion of Orange Flavor in Yeast Cells Instead of menthol as the flavor, orange flavor is used as the flavor, and the orange flavor is included in the yeast cells. The rate (%), surface content rate (%), and inclusion rate (%) were determined.

(1) Preparation of orange flavor inclusion yeast powder (1-1) For yeast A and yeast C, yeast from which cell contents in the cells have been removed by the method described in Example 2 (1-1) An aqueous dispersion (10% dry solids) was prepared containing (unmodified method).
Next, 450 g of each dispersion was heated to 60° C., and 25 g of orange fragrance (manufactured by San-Ei Gen FFI Co., Ltd.) containing D-limonene (logP: 4.38) as a main fragrance component was added. The mixture was stirred at 3000 rpm for 2 hours. Further, 60 g of a 50% lactose-containing aqueous solution heated to 60° C. was added and mixed. Next, these mixed solutions are spray-dried under the conditions of an inlet temperature of 150° C. and an outlet temperature of 90° C. to obtain powdery orange flavor clathrate yeast powder (abbreviated as “OR flavor clathrate yeast”). 90 g was obtained (Comparative Example 4: derived from yeast A, Comparative Example 5: derived from yeast C). Theoretically, the total powder should be 100 g, which means that 10 g was not recovered during the spray drying process.

<Composition of flavor inclusion yeast>
Yeast cell wall: 45 parts by mass Orange flavor: 25 parts by mass
Excipient (lactose): 30 parts by mass
Whole: 100 parts by mass

(1-2) For yeast A and yeast C, the cell contents in the cells were removed using improved method 2 described in Experimental Example 3 (1) (1-2) instead of the unimproved method. It was obtained as a yeast cell wall fraction.
Then, 90 g of powdery orange flavor inclusion yeast powder (OR flavor inclusion yeast) was obtained according to the method described in (1-1) above (Example 5: derived from yeast A, Example 6: derived from yeast C).

(2) Quantitation of Orange Flavor Content Encapsulated in Yeast Cells
(2-1) Quantification of total content
0.04 g of the OR flavor inclusion yeast prepared above was suspended in 20 mL of ultrapure water and subjected to ultrasonic treatment for 5 minutes to uniformly disperse the bacterial cell powder in the pure water. Then, 0.5 mL of 10% 1-octanol solution (solvent: acetone) and 29.5 mL of acetone were added to prepare a test solution. The solution was quantified for D-limonene content by GC. The quantification of the orange flavor was converted to the D-limonene equivalent. The quantification of D-limonene was performed using a calibration curve prepared in advance using D-limonene as a standard and 1-octanol as an internal standard (calibration curve method) (same below).

(2-2) Determination of Surface Content 0.5 g of the OR flavor inclusion yeast prepared above was suspended in 50 mL of n-hexane and stirred with a stirrer for 30 seconds. Then, the suspension was filtered with filter paper, 1 mL of 10% 1-octanol solution (solvent: acetone) was added to the filtrate as an internal standard, 49 mL of acetone was added to prepare a test solution, and the content of D-limonene was quantified by GC. bottom.

(3) Results Table 6 shows the results of measuring the inclusion rate of orange flavor (D-limonene) in orange flavor clathrate yeast.

As shown in Table 6, both yeasts A and C had their cell contents removed using the improved method described above (Examples 5 and 6) compared to yeast prepared by the unimproved method. As a result, it was confirmed that more orange flavor (D-limonene) can be included in the cells. As shown in Comparative Examples 4 and 5, although the inclusion rate of the flavor varies depending on factors on the yeast side, by removing the contents of the fungal cells using the improved method, the variation in the inclusion rate caused by the yeast can be reduced. It was also confirmed that it can be almost uniform. From the above results, similarly to menthol, orange flavoring (D-limonene) was also found to have a median diameter (D ₅₀ ) in the range of 1 to 10 μm by adjusting the particle size distribution of the yeast to be included. It was found that the encapsulation rate of perfume can be improved regardless of species.

Experimental Example 6 Influence of Inclusion Rate in Yeast Cells Based on Differences in Flavor Using multiple perfume ingredients with different hydrophobicities (see Table 6), the inclusion rate in yeast cells was measured and evaluated. Octanol/water partition coefficient (Pow) was used as an index indicating the hydrophobicity of perfume ingredients.

Specifically, using yeast C, 60 parts of an aqueous yeast suspension (dry solid content: 10%) prepared in the same manner as in Experimental Example 2 (1) (1-1) is described in Table 7. 40 parts of each flavor component was added, and the mixture was stirred at 3000 rpm for 60 to 120 minutes to obtain a flavor clathrate yeast solution. The solution was observed at 600x using an inverted microscope. When the flavor penetrates into the cells through the cell walls and is included, the yeast cells swell, and the outer edges of the cell walls are clearly observed (see, for example, FIG. 4(a)). On the other hand, in the case of yeast in which the flavor is not sufficiently clathrated, the cells do not swell and the outer edge is obscured (see, for example, FIG. 4(b)).

After 60 minutes and 120 minutes from the stirring, the above-mentioned microscopic observation was carried out, and the adequacy of the encapsulation property of the perfume was evaluated according to the following criteria.
[Evaluation of inclusion property of perfume]
○: The outer edge was clearly observed 60 minutes after stirring (inclusion confirmation)
△: The outer edge was unclear 60 minutes after stirring, but was clearly observed after 120 minutes (inclusion confirmation)
x: The outer edge is unclear even after 120 minutes from stirring (unconfirmed inclusion).

Table 7 shows the results.

From this result, acids, aldehydes, esters, and alcohol-based fragrance ingredients with an octanol/water partition coefficient (Pow) of 1.5 to 4.8 in logP value permeate into the yeast cells and are clathrated in the cells. It was confirmed that On the other hand, no inclusion in the yeast cells was observed for perfume components with a Pow of less than 1.5 or more than 4.8 in terms of logP value.
From this, it is considered that the inclusion property of the flavor in the yeast cells is strongly influenced by the hydrophobicity (log P value) of the flavor to be included.

From Experimental Example 6 above, as the perfume component used in the preparation of the perfume inclusion yeast, at least one perfume component selected from acids, aldehydes, esters, and alcohols having a logP value of 1.5 to 4.8 is used. confirmed to be favorable. In addition, from the above Experimental Examples 2 to 5, regardless of the type of yeast, as the yeast to clathrate the fragrance, by using yeast having a particle size distribution with a median diameter (D ₅₀ ) in the range of 1 to 10 μm, It was confirmed that the clathration amount (clathration rate) of the fragrance component into the body can be uniformly increased.

Claims (7)

A powdered flavoring agent for food and drink, in which a hydrophobic flavoring agent is included in yeast cells from which cell contents have been removed,
A powdered flavoring agent for food and drink, wherein the yeast cells have a median diameter D50 of 1 μm to 10 μm in a volume-based particle size distribution measured on the aqueous suspension using a laser diffraction/scattering particle size distribution analyzer. The powdered flavorant for food and drink according to claim 1, wherein the hydrophobic flavorant has an octanol/water partition coefficient (logP) of 1.5 to 4.8. The hydrophobic perfume is menthol, l-menthol, dl-menthol, 3-l-menthoxypropane-1,2-diol, isopulegol, neoisopulegol, isoisopulegol, neoisopulegol, neomenthol, isomenthol , neoisomenthol, citronellol, linalool, peppermint oil, peppermint oil, menthyl lactate, isopulegol, menthyl succinate, α-pinene, β-pinene, sabinene, myrcene, D-limonene, linalool, and γ-terpinene The powdered flavoring agent for food and drink according to claim 1 or 2, which is at least one selected. The powdered flavoring agent for food and drink according to any one of claims 1 to 3, which is a flavoring agent for chewing gum. Subjecting the yeast cells to a process of eluting the cell contents in the cells and a fractionation process,
A step of fractionating a fraction having a median diameter D50 of 1 μm to 10 μm in the volume-based particle size distribution measured for the aqueous suspension using a laser diffraction/scattering particle size distribution analyzer to obtain a yeast cell wall fraction. and contacting the yeast cell wall fraction obtained in the above step with a hydrophobic flavor;
A method for preparing a powder flavoring for food and drink, comprising: A preparation method according to claim 5, wherein the hydrophobic perfume is a perfume having an octanol/water partition coefficient (logP) of 1.5 to 4.8. A method for improving inclusion of a hydrophobic flavoring agent in a yeast cell wall fraction, comprising:
The yeast cells are adjusted so that the median diameter D50 in the volume-based particle size distribution measured for the aqueous suspension using a laser diffraction/scattering particle size distribution analyzer is 1 μm to 10 μm. and how.

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