JP5608344B2 - Yeast having immunostimulatory ability and food or feed - Google Patents

Description

  The present invention relates to a yeast that is excellent in safety and can exhibit a high immunostimulatory ability even when ingested in the form of bacterial cells, and a food or feed containing the same.

Immunity is a mechanism in which a living body protects itself from attacks on the living body by viruses, bacteria, cancer cells, and the like that have entered from outside. Many types of cells are involved in immunity, but macrophages are ubiquitous in all animal species, and are involved in all stages of the immune response, particularly in the early stages. Therefore, its importance is recognized.
It is known that various diseases such as cancer, infectious diseases, and allergic symptoms are induced when immunity is lowered. On the other hand, when immunity is activated, various effects such as suppression of carcinogenesis, anticancer activity, anti-infection, anti-allergy, and recovery of physical condition rhythm and homeostasis can be expected.

Therefore, various compounds and microorganisms having an immunostimulatory action are provided for the purpose of improving the health condition of consumers. For example, those using a hydrolyzate of lactoferrin (see Patent Document 1), chitin (see Patent Document 2), trehalose (see Patent Document 3), fucoidan (see Patent Document 4), plant-derived Those using substances (see Patent Document 5), those using peptides (see Patent Document 6) and interleukins (see Patent Document 7), those using nucleic acids (see Patent Document 8), glutathione (see Patent Document 9), Microorganisms such as amino acids (see Patent Document 10), polylysine (see Patent Document 11), enterobacteria (see Patent Document 12), lactic acid bacteria (see Patent Document 13), Enterococcus bacteria (see Patent Document 14), etc. And the like to be used.
In recent years, the immunostimulatory effect of β-glucan has been attracting attention. For example, β-glucan is purified from yeast or mushrooms, and an immunostimulatory method using the β-glucan is proposed (Patent Documents 15 to 16). reference).

  However, in order to activate consumer immunity using the compound having the immunostimulatory action, it is necessary to purify and administer the compound in a large amount, which requires many working steps, labor and cost. . In addition, among the microorganisms having the immunostimulatory action, there are those whose properties are not clear, and there are those that are not suitable for food use. In addition, when the microorganism having immunostimulatory ability itself cannot be directly used for food production (fermentation, etc.), it is necessary to add it separately to the food in order to confer immunostimulatory ability to the food. there were.

  Here, among the genes on the yeast chromosome, yeast having immunostimulatory ability as a result of disruption of one or more genes among the group consisting of MCD4 gene, GAS1 gene, and CWH41 gene Is disclosed (see Patent Document 17). According to the yeast disclosed in Patent Document 17, even when the yeast is administered as it is without purifying β-glucan from the yeast as in the techniques described in Patent Documents 15 and 16, Immunity can be activated.

However, in the yeast disclosed in Patent Document 17, the specific gene is destroyed by gene recombination technology. In recent years, with the development of genetic engineering technology, a number of genetically modified foods have been approved or sold after their safety has been confirmed. From the consumer's perspective, the safety of genetically modified foods Anxiety is still high. Therefore, at present, it is practically difficult to use the yeast disclosed in Patent Document 17 for food.
Furthermore, it was revealed that the yeast disclosed in Patent Document 17 grows very slowly in a medium with low osmotic pressure, as expected from the lack of genes related to the structure of the cell wall. Therefore, in the yeast culture, for example, sugar is added to the medium to increase the osmotic pressure of the medium so as not to apply a load to the cell wall that has become brittle, and the medium and the solution in the yeast cell membrane There was a problem that the osmotic pressure of the solution had to be made isotonic and the culture cost increased. In addition, when the yeast is used for fermentation of foods such as bread, for example, the fermentation conditions are limited, and when the sugar is added to make the fermentation solution isotonic, the flavor of the food changes. There was a problem, such as being loose.

  On the other hand, the mutagenesis method is a method of causing a mutation in DNA more frequently than natural mutation by a mutagenesis factor such as a mutagenesis agent, ultraviolet irradiation, and radiation irradiation. Unlike the gene recombination technique, the mutagenesis method does not introduce a base sequence other than the base sequence inherent in the food. Therefore, foods whose genetic traits have been changed by the mutagenesis method are accepted by consumers as being relatively safe even in the present situation.

  Therefore, it is excellent in safety, can exhibit high immunostimulatory ability even if it is ingested as bacterial cells, is easily available and inexpensive, can be directly used for food production, and can be cultured even at low osmotic pressure. In addition, microorganisms such as yeast having a low agglutination property during culture and a high yield to sugar have not been provided yet, and there is a strong demand for prompt provision thereof.

JP-A-5-178759 JP-A-6-271470 JP 2003-81839 A JP 2001-181303 A Japanese Patent Laid-Open No. 6-56785 Japanese Patent No. 2873434 JP 2002-3395 A Japanese Patent No. 2529605 JP 11-49696 A JP 2000-281571 A JP 2003-128589 A JP-A-6-56678 JP-A-7-228536 Japanese Patent Laid-Open No. 11-92389 JP 2001-354570 A JP 2003-183176 A JP 2006-75039 A

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention is excellent in safety, can exhibit high immunostimulatory ability even when ingested as a bacterial cell, is easily available and inexpensive, can be directly used for food production, and has low osmotic pressure. However, an object is to provide a yeast that can be cultured, has a low cohesiveness during culture, and has a high yield to sugar, and a food or feed containing this yeast.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, when a mutation is introduced into the yeast chromosome by a mutagenesis method using ethyl methane sulfonic acid to a yeast that is currently widely used in food production, it is possible to obtain a yeast having a low cell wall mannan. This yeast has β cell glucan exposed on the surface of the cell due to the small amount of mannan on the cell wall. Therefore, it is not necessary to purify β glucan from this yeast. The yeast showed significant immunostimulatory capacity; the yeast was unexpectedly highly resistant to low osmotic pressure, despite the low cell wall mannan, and also during culture It has been found that it can be suitably used for the production (cultivation) of yeast itself and the production of foods and feeds containing this yeast because of its low cohesiveness and high sugar yield.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> Cell wall has few mannans, has immunostimulatory ability, can be grown on YPD liquid medium with osmotic pressure of 300 m Osm, yield to sugar is 75% by mass or more, and genome number is diploid or more It is yeast characterized by being.
<2> The yeast according to <1>, wherein the yeast is non-uracil-requiring.
<3> The yeast according to any one of <1> to <2>, wherein the immunostimulatory ability is a macrophage activating ability.
<4> The yeast according to any one of <1> to <3>, which has acquired immunostimulatory ability by a mutagenesis method.
<5> The yeast according to any one of <1> to <4>, wherein the mutagenesis method is a method using any one of a mutagenesis agent, ultraviolet irradiation, and irradiation.
<6> The yeast according to any one of <1> to <5>, wherein the yeast is not genetically modified.
<7> The yeast according to any one of <1> to <6>, wherein the parent strain of the yeast having a genome number of diploid or more is Saccharomyces cerevisiae .
<8> Saccharomyces cerevisiae FERM P-21728, Saccharomyces cerevisiae FERM P-21729, or Saccharomyces cerevisiae FERM P-21730.
<9> A food or feed containing the yeast according to any one of <1> to <8>.

  According to the present invention, the conventional problems can be solved, the object can be achieved, the safety is excellent, and even if the cells are ingested as they are, they can exhibit a high immunostimulatory ability and are easily available. In addition, it can be used directly in the production of food, can be cultured even at low osmotic pressure, has low coagulation during culture, has a high yield to sugar, and a food containing the yeast or Feed can be provided.

FIG. 1 is a microscopic image of mutants 1-4. FIG. 2 is a diagram showing the results of a macrophage activation ability test of mutants 1 to 4. FIG. 3 is a microscopic image of mutants 6-8. FIG. 4 is a diagram showing the results of a macrophage activation ability test of mutants 6-8. FIG. 5A is a diagram showing the results of a macrophage activation ability test of practical strains and mutants 6-8. FIG. 5B is a diagram showing the results of LPS-stimulated macrophage activation ability used as positive controls in the macrophage activation ability test of the practical strains and mutants 6-8. FIG. 6A is a diagram showing the degree of growth of the mutant 4 and mutants 6 to 8 cultured in a uracil-added medium. FIG. 6B is a diagram showing the degree of growth of the cells obtained by culturing the mutant 4 and the mutants 6 to 8 in a medium not added with uracil. FIG. 7 is a diagram illustrating the aggregation properties of the mutant 4 and the mutants 6 to 8. FIG. 8 is a diagram showing bread produced by a wild type diploid strain, a practical strain E, and mutants 6-8.

(yeast)
The yeast of the present invention has a low cell wall mannan, has an immunostimulatory ability, can grow on a YPD liquid medium having an osmotic pressure of 300 m Osm, has a yield of saccharides of 75% by mass or more, and has 2 genomes. It is characterized by being more than a ploidy.

<Mannan>
In general, mannan is one of the main components constituting the cell wall of yeast and is a sugar chain mainly composed of D-mannose. The mannan exists as a mannan protein bound to a protein. In the cell wall of yeast, a β-glucan layer is formed along the outer peripheral surface of the cell membrane, and the mannan protein is bound to the β-glucan via a glycolipid called GPI (glycosylphosphatidylinositol) anchor. As a result, mannan is arranged on the outermost layer of the cell wall (that is, the cell surface) so as to cover the β-glucan. On the other hand, in the yeast of the present invention, β-glucan is exposed on the cell surface instead of mannan due to the small amount of mannan on the cell wall.

  The reason for the low amount of mannan in the cell wall in the yeast is not clear, but for example, at least one of mannan, mannan protein, and GPI anchor is not biosynthesized, biosynthesized but not transported outside the cell membrane, Possible reasons include deletion or mutation at the binding site.

-Mannan detection-
In the yeast, the amount of mannan in the cell wall is small, for example, ConA (Concanavalin A) that specifically binds to an antibody specific for mannan, D-mannose or methyl-α-D-mannopyranoside in mannan, and the like. It can be confirmed using lectins and the like.
Specifically, the antibody or ConA is labeled with, for example, a fluorescent dye such as FITC (Fluorescein isothiocyanate), and the binding of the antibody or ConA to the cell surface is detected by fluorescence (hereinafter referred to as “mannan”). Sometimes referred to as "staining"). In this case, if fluorescence derived from the binding of the antibody or ConA is not detected on the cell surface, it can be determined that the mannan on the cell wall is small.
The detection of the fluorescence may be performed visually under a fluorescence microscope, for example, may be quantitatively performed using an apparatus such as a flow cytometer or a fluorescence plate reader.

Moreover, in the said yeast, it can be investigated by quantitating mannan content in a cell wall that there are few mannans in a cell wall, for example. Although there is no restriction | limiting in particular as a method of quantifying mannan content, For example, it can quantitate with the following procedures.
That is, first, cells are disrupted with glass beads, a cell wall fraction is prepared by centrifugation, and lyophilized. Next, sulfuric acid hydrolysis is performed according to the method of Dallies et al. (Yeast, 14, 1998, p. 1297-1306), and the supernatant after neutralization is lyophilized, and reZEXTM RPM-Monosaccharide Phenomenex (ShimadzuGLC LTD) and the like The content of mannan in the cell wall can be determined by analyzing by HPLC using a column and determining the ratio of glucose and mannose.

  The content of mannan in the cell wall is not particularly limited as long as β-glucan is reduced to such an extent that it is exposed on the cell surface, and it is more preferable that the cell wall does not have mannan. Hereinafter, a yeast with a small amount of mannan and β-glucan exposed on the surface of the cell may be referred to as “glucan-exposed yeast”.

-Β-glucan-
The β-glucan is one of the main components constituting the cell wall of yeast, and is a sugar chain formed by binding glucose with β1-3 type.
The average molecular weight of β-glucan and the modification mode are not particularly limited as long as they have immunostimulatory ability, and may vary depending on the type of yeast and culture conditions.

  In the yeast, the method for confirming that β-glucan is exposed on the cell surface is not particularly limited and can be appropriately selected according to the purpose, for example, an antibody specific for β-glucan, This can be confirmed using a fluorescent reagent such as aniline blue. Moreover, it can also confirm by observing the beta glucan fiber on the surface of a microbial cell with an electron microscope.

<Immunostimulatory ability>
The immunostimulatory ability is the ability to activate innate immunity. The innate immunity means a mechanism that is inherently provided in the living body of the immunity of the living body and functions when the antigen is removed from the living body even if it has not been exposed to the antigen in advance. The antigen is not particularly limited as long as the effects of the present invention are not impaired, and examples thereof include foreign antigens such as viruses and bacteria, and endogenous antigens such as cancer cells.

  The immunostimulatory ability is not particularly limited as long as the innate immunity can be activated, and can be appropriately selected according to the purpose. For example, phagocytic cells (macrophages, monocytes, neutrophils, dendritic cells), Examples thereof include the ability to activate antigen-presenting cells (macrophages, dendritic cells), natural killer (NK) cells, polymorphonuclear leukocytes (eosinophils, basophils, mast cells) and the like. Among these, the macrophage activation ability is preferable because it is widely present in various organisms and has a high effect of eliminating antigens.

  The reason why the yeast has the immunostimulatory ability is considered to be that there are few mannans on the cell wall and β-glucan having the immunostimulatory ability is exposed on the surface of the cells.

-Evaluation of immunostimulatory ability-
There is no restriction | limiting in particular as a method of evaluating that the said yeast has the said immunostimulatory ability, It can select suitably from the conventionally well-known evaluation methods according to the kind of the said cell involved in innate immunity.
For example, the macrophage activation ability can be evaluated by stimulating the macrophage by contacting the macrophage with the yeast in a medium or a buffer, and producing a cytokine (for example, TNF) before and after the stimulation. -Α (Tumor Necrosis Factor-α) and the like can be measured.

<Tolerance to osmotic pressure>
In general, some microorganisms including yeast and cell walls provided in plants have extremely important functions such as resistance to osmotic pressure and maintenance of cell morphology.
On the other hand, since the yeast of the present invention has a small amount of mannan on the cell wall as described above, there has been a concern that the resistance to osmotic pressure is lost. However, it has become clear from the examples described later that the yeast produced by the present inventors has resistance to osmotic pressure unexpectedly.

-Evaluation of resistance to osmotic pressure-
The index indicating that the yeast of the present invention is resistant to low osmotic pressure is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Can be grown on YPD liquid medium of 300 m Osm. However, the YPD liquid medium has a composition of 1% by mass yeast extract, 2% by mass peptone, and 2% by mass glucose. Moreover, as yeast extract used for said YPD liquid culture medium, the thing made by Oriental Yeast Industry Co., Ltd. is used, as a peptone, the thing made by Nippon Seiyaku Co., Ltd. is used, and domestic chemical (stock) is used. ) Shall be used. The osmotic pressure can be adjusted with, for example, non-fermentable saccharides such as sorbitol, NaCl, and the like. The osmotic pressure can be measured by, for example, an osmometer F-2000 (manufactured by Roebling).

In the present invention, “proliferating” means that the yeast cultured in a YPD agar medium (1% by weight yeast extract, 2% by weight peptone, 2% by weight glucose, 2% by weight agar) is a YPD liquid that is a hyperosmotic medium. When 1 platinum ear inoculation is carried out on the medium and shake culture is performed overnight under appropriate conditions, it means that the yield to sugar is 75% by mass or more. The yield to sugar refers to the wet mass of the yeast recovered per gram of sugar.
For example, after the yeast suspension obtained in the culture was washed with distilled water, the dry (DM) mass of the yeast was measured, and the yield of sugar was recovered per 1 g of sugar. Examples include a method of calculating wet mass (with a water content of 67% by mass) (g).

  Since the yeast can grow on a YPD liquid medium with an osmotic pressure of 300 m Osm, for example, it is not necessary to add sugar or the like to increase the osmotic pressure of the medium, which is advantageous in that the culture cost can be reduced. It is. In addition, when the yeast is added to food, since the cells are not destroyed even after washing with a low osmotic pressure solution after cultivation, the recovery rate of viable bacteria can be increased and the production efficiency of food can be increased. This is also advantageous in that

<Shares>
Whether or not the yeast of the present invention has an auxotrophy is not particularly limited and can be appropriately selected according to the purpose, but the non-nutrient-requiring strain can reduce the culture cost. preferable. There is no restriction | limiting in particular as said nutrition, According to the objective, it can select suitably, For example, uracil etc. are mentioned.

-Uracil content in the medium-
When screening is performed, it is determined whether or not the yeast is uracil-requiring. The YPD liquid medium to be used contains 0.3% by mass or more of uracil with respect to the glucose content. I will do it. That is, when the content of glucose in the YPD liquid medium is 2% by mass, the content of uracil in the YPD liquid medium is 6 × 10 ⁻³ % by mass or more.
If the yeast to be screened has other requirements, the YPD liquid medium to be used should be screened with a sufficient amount of the required substance.

<Type of yeast>
There is no restriction | limiting in particular as said kind of yeast, According to the objective, it can select suitably, For example, baker's yeast, beer yeast, wine yeast, sake yeast, miso soy sauce yeast, etc. are mentioned. Among these, baker's yeast is preferable.

  The yeast is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include Saccharomyces, Zygosaccharomyces, Yarrowia, Williopsis, Examples include the genus Torulaspora, the genus Candida, the genus Rhodotorula, the genus Pichia and the like.

Species of the yeast, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Zygosaccharomyces rouxii, Candida saitoana, Candida tropicalis, Yarrowia lipolytica, Torulaspora delbrueckii, Candida sake, Candida tropicalis, Candida utilis, Pichia anomala, Williopsis saturnus, Saccharomycopsis fibligera, Rhodotorula glutinis, Pichia farinos , And the like.
Among these, Saccharomyces cerevisiae and Saccharomyces pastorianus are preferable, and Saccharomyces cerevisiae is particularly preferable.

As the Saccharomyces cerevisiae, not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably Saccharomyces cerevisiae FERM P-21728, Saccharomyces cerevisiae FERM P-21729, and Saccharomyces cerevisiae FERM P-21730, immunopotentiating capability Saccharomyces cerevisiae FERM P-21729 and Saccharomyces cerevisiae FERM P-21730 are more preferable, and the production suitability is good because the aggregation property during production (culture) is low and the yield to sugar is high. In Saccharomyces cerevisiae P-21 729 is particularly preferred.
Saccharomyces cerevisiae FERM P-21728, Saccharomyces cerevisiae FERM P-21729, and Saccharomyces cerevisiae FERM P-21730 are stocks produced by the present inventors. Has been.

<Culture method>
The yeast culture method is not particularly limited and may be appropriately selected from conventionally known methods according to the purpose. Examples thereof include batch culture and fed-batch culture.
The culture can be suitably performed using a jar fermenter, and the conditions in the jar fermenter at this time are not particularly limited and can be appropriately determined. For example, the culture temperature is 28 ° C. to 33 ° C. The culture time is about 1 hour to 120 hours, the pH is about pH 4 to pH 7, the aeration amount is about 0 vvm to 5 vvm, and the stirring speed is about 100 rpm to 700 rpm.

-Fed-batch culture-
The fed-batch culture method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, after using 300 g to 500 g of the seed yeast cultured in the YPD medium, The total addition amount is sugar amount (sucrose conversion) 1,000 g to 2,000 g, urea 100 g to 200 g, monosodium phosphate 20 g to 40 g, etc. It is done.

<Manufacturing method>
The method for producing a yeast having a genome number of diploid or more is not particularly limited and can be appropriately selected according to the purpose. For example, a yeast that can be used for food is used as a parent strain, A method of producing a mutation on a chromosome by a mutagenesis method, a method in which a yeast that can be used for food is used as a parent strain, and a mutation is caused on the chromosome by spontaneous mutation in this parent strain. Examples include a method of subjecting strains to mutagenesis.
There is no restriction | limiting in particular as a parent strain | stump | stock which can be used for the said food use, Although it can select suitably according to the objective, For example, the yeast illustrated in the item of <type of yeast> mentioned above can be utilized.
The parent strain of yeast having a genome number of diploid or more may be a haploid strain, and a diploid can be obtained by crossing the haploid strain.

-Mutagenesis method-
The mutagenesis method means a method of causing a mutation in DNA at a higher frequency than natural mutation by a mutagenesis factor such as a mutagenesis agent, ultraviolet irradiation, or radiation irradiation. By using the mutagenesis method, mutation can be caused on the chromosome without introducing a base sequence other than the base sequence originally possessed by the parent strain.
There is no restriction | limiting in particular as said mutagenic agent, According to the objective, it can select suitably, For example, ethylmethanesulfonic acid, nitrosoguanidine, 5-bromouracil etc. are mentioned.
Examples of the radiation include X-rays, α-rays, β-rays, γ-rays, and particle beams.
The mutagenesis method is advantageous in that it can be used in foods because it is highly safe unlike genetic recombination because mutation occurs in the bases inherent in yeast that can be used for food applications.

There are no particular limitations on the gene that causes mutation by the mutagenesis method or the natural mutation, as long as the parent strain can acquire immunostimulatory ability. For example, RPS12, MRS6, GPB1, RAD17, NDD1, SCP1, MCD4, GAS1 , CWH41 and other genes. In addition, base sequences on chromosomes other than these genes may be mutated.
The type of mutation is not particularly limited as long as the parent strain can acquire immunostimulatory ability, and examples thereof include base substitution, insertion, deletion, inversion and the like.

--Primary screening of -1 ploidy--
The method for screening a strain having a low mannan from the haploid mutant obtained by the mutagenesis method is not particularly limited and can be appropriately selected according to the purpose. For example, SDS of a strain having a low mannan is used. The method using the sensitivity with respect to is mentioned. By comparing the growth of the haploid mutant strain in the SDS-added medium and the SDS-free medium, and selecting a strain that grows slowly or kills in the SDS-added medium, a strain with less mannan (glucan) Exposed yeast).

-Secondary screening of -1 ploidy-
As a method for screening a haploid strain of glucan-exposed yeast, it is preferable to select a strain with less mannan by further staining the haploid mutant obtained in the primary screening with the mannan.

-Third screening of haploids-
The method for screening a strain having immunostimulatory ability from the glucan-exposed yeast haploid strain obtained by the secondary screening is not particularly limited and can be appropriately selected according to the purpose. An immunostimulatory evaluation method can be used.

-Production of glucan-exposed yeast diploid strain-
The glucan-exposed yeast diploid strain is a parent strain obtained by crossing the haploid-exposed glucan-exposed yeast haploid strain obtained in the haploid tertiary screening with a haploid wild strain. Can be obtained as its descendants.

--Glucan-exposed yeast haploid strain having immunostimulatory ability--
The glucan-exposed yeast haploid strain is not particularly limited as long as it has an immunostimulatory ability and has few mannans, and can be appropriately selected according to the purpose. A strain obtained by production of a body strain is preferable, and Saccharomyces cerevisiae FERM P-21355 is more preferable.
The Saccharomyces cerevisiae FERM P-21355 is deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center.

--Manufacture of parent strains of --2
The parent strain of the glucan-exposed yeast diploid strain can be obtained by crossing the glucan-exposed yeast haploid strain with a haploid wild strain. There is no restriction | limiting in particular as the said hybridization method, According to the objective, it can select suitably, For example, a conventional method can be used.
Here, the trait of the glucan-exposed yeast haploid strain is a strain that requires uracil and is low in mannan (hereinafter sometimes referred to as “uracil (−) mannan (−) strain”). When the ploidy wild strain is a strain that does not require uracil and has normal mannan (hereinafter, sometimes referred to as “uracil (+) mannan (+) strain”), these two were crossed. Ploidy strains are heterozygous in uracil auxotrophy and mannan traits. In this case, the diploid strain is spore-separated, and after obtaining a haploid strain again, the haploid strain after separation of the spore having a desired trait is crossed to obtain uracil requirement, and A homozygous diploid strain can be obtained for mannan traits.

--Primary screening of diploid--
The desired trait of the glucan-exposed yeast diploid strain is preferably uracil non-required from the viewpoint that the culture cost can be reduced. Therefore, in the primary screening, a uracil non-required strain and a low amount of mannan (hereinafter sometimes referred to as “uracil (+) / mannan (−) strain”) from the haploid strain after spore isolation. It is preferable to screen.
The primary screening method is not particularly limited and can be appropriately selected according to the purpose. For example, after selecting a strain capable of growing in a medium not containing uracil using a medium not containing uracil. Furthermore, similarly to the above-described primary screening for haploids, a method of selecting strains that grow slowly or die in the above-described SDS-added medium using the sensitivity to SDS can be used.
The medium not containing uracil is not particularly limited and may be appropriately selected depending on the intended purpose. For example, SD medium (0.67% by mass Yeast Nitrogen Base (W / O amino acid), 2% by mass) Glucose, 2 mass% agar) and the like.

--- Production of uracil non-required glucan-exposed yeast diploid--
The method for obtaining a diploid strain from a haploid of the uracil (+) mannan (-) strain is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the uracil (+) It can be obtained by confirming the haploid mating type of the mannan (−) strain and performing mating matrix determination.
The method for confirming the mating type is not particularly limited and can be appropriately selected depending on the purpose. For example, a haploid strain obtained by the primary screening and a strain whose known mating type is known. Examples include a method of co-culturing and visually confirming aggregation.

--Secondary screening of diploid--
In order to select a glucan-exposed yeast diploid strain having less mannan from the uracil (+) / mannan (−) diploid obtained by the mating matrix determination, a secondary screening is performed. It is preferable.
There is no restriction | limiting in particular as the method of the said secondary screening, According to the objective, it can select suitably, For example, the method similar to the secondary screening of the haploid mentioned above can be used.

--Evaluation of macrophage activation ability of diploid--
The method for evaluating the immunostimulatory ability of the diploid strain of glucan-exposed yeast obtained by the secondary screening of the diploid is not particularly limited and can be appropriately selected depending on the purpose. A method similar to the haploid tertiary screening can be used.

<Examination of manufacturing aptitude>
Since the number of genomes of the yeast is diploid or more, the yeast is a stable yeast that can solve the problem of haploids, that is, the problem that the properties of haploids change due to contamination because they have mating ability. It is advantageous in production because it has excellent growth ability compared to haploid, has a low aggregation property compared to haploid, is non-nutritive and can reduce culture costs. It is.
A method for examining the production suitability of the glucan-exposed yeast diploid is not particularly limited and can be appropriately selected according to the purpose. For example, the yield to sugar, auxotrophy, aggregation property, etc. are used as indicators. The method to consider is mentioned.

-Yield to sugar-
The sugar yield is as described above, and when the sugar yield is high, the yield of the yeast is high, that is, the growth ability is high.
There is no restriction | limiting in particular as said sugar yield, Although it can select suitably according to the objective, 75 mass% or more is preferable, 80 mass% or more is more preferable, 95 mass% or more is still more preferable. Within the preferable range, it is advantageous in that the production cost can be reduced.

-Nutritional requirements-
As described above, the auxotrophy of the yeast is preferably non-nutritional as it can reduce the culture cost.
A method for confirming whether or not the yeast is non-nutrient-requiring is not particularly limited and can be appropriately selected according to the purpose. For example, the yeast is inoculated into a medium not added with uracil. And a method for confirming that growth is possible in a medium not added with uracil.

-Aggregation-
It is preferable that the yeast does not have aggregability. If the yeast has aggregability, there is a problem in that the separator is clogged in the manufacturing process.
The method for confirming whether or not the yeast has aggregability is not particularly limited and may be appropriately selected depending on the intended purpose. For example, after culturing overnight under appropriate conditions using a YPD liquid medium And a method of confirming the yeast by observing it with an optical microscope.

<Application>
The use of the yeast is not particularly limited and can be appropriately selected according to the purpose. However, the use as a food material, feed, feed, etc. used by adding to food is preferable, and the use as the food material. Is particularly preferred. Moreover, the said yeast can be used suitably for manufacture of foodstuffs, such as fermentation of bread and beer, for example. By using the yeast of this invention, the food which has immunopotentiation ability, the feed which has immunopotentiation ability, the feed which has immunopotentiation ability, etc. are obtained. At this time, the yeast may be used in a state in which the cells are not crushed, may be used in a crushed state, may be used in a dried state, It may be used in an undried state.
There is no restriction | limiting in particular as said crushing method, According to the objective, it can select suitably, For example, physical crushing processes, such as a dyno mill, and a chemical crushing process may be sufficient.

  The yeast is not particularly limited as long as it has innate immunity. For example, humans, animals other than humans (horse, cattle, pigs, sheep, goats, camels, llamas, livestock, mice, rats, guinea pigs, etc. , Laboratory animals such as rabbits, poultry such as chickens, ducks, turkeys and ostriches, red sea bream, sea bream, flounder, flounder, yellowtail, hamachi, hiramasa, tuna, shimaji, ayu, salmon trout, trough puffer, eel, loach, catfish Fish, shrimp, black tiger, crustacean such as tiger shrimp, crab, etc., shellfish such as abalone, turban shell, scallop and oyster, pet animals such as dog and cat).

(Food or feed)
The food or feed of the present invention is characterized by containing the yeast.
The food is not particularly limited and may be appropriately selected depending on the intended purpose. For example, confectionery such as bread, biscuits and scones, hospital food, liquid food, processed fishery products, noodles, seasonings, beer And beverages such as juices, health foods and health drinks.
There is no restriction | limiting in particular as said feed, According to application object, it can select suitably. There is no restriction | limiting in particular as said application object, For example, the application object illustrated by the item of the use of the said yeast is mentioned.
The food or feed has high immunostimulatory ability by containing the yeast having high immunostimulatory ability.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

(Example 1: Yuploid yeast having immunostimulatory ability)
<Production of glucan-exposed yeast haploid strain>
-Selection of natural mutants-
As a parent strain to be subjected to the mutagenesis method, a uracil-requiring mutant strain T-21U of T-21 strain (produced by Oriental Yeast Co., Ltd.), which is a haploid of practical baker's yeast, was used.
The T-21U strain was prepared by culturing the T-21 strain in a YPD liquid medium, then containing a medium containing 0.7-fluoroorotic acid (0.7% by mass YEAS NITRogen BASE (manufactured by DIFCO)), 2% by mass glucose, 0. 1% by mass 5-fluoroorotic acid, 0.05% by mass uracil, 2% by mass agar), and the strain was selected as a natural mutant by growing it at 30 ° C. for 3 days. .

-Mutagenesis method-
Uracil-containing YPD liquid medium (1% by weight yeast extract (made by Oriental Yeast Co., Ltd.), 2% by weight peptone (made by Nippon Pharmaceutical Co., Ltd.), 2% by weight glucose (made by Kokusan Kagaku Co., Ltd.), 6 × 10 ^The parent strain (T-21U strain) was cultured overnight using ^-3 mass% uracil (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter the same). The cultured cells were dispensed into two Eppendorf tubes, collected by centrifugation. The supernatant was removed, washed twice with sterilized water, and the cells were suspended in 0.1 M (mol / L, hereinafter the same) phosphate buffer (pH 7.0). 30 μL of ethyl methanesulfonate (EMS) (manufactured by Wako Pure Chemical Industries, Ltd.) was mixed and incubated at 30 ° C. for 1 hour with shaking. The cells were collected by centrifugation, the supernatant was discarded, and resuspended in 200 μL of 5% by mass sodium thiosulfate. The suspension was transferred to a new tube, centrifuged, and washed with 200 μL of 5% by mass sodium thiosulfate twice. The cells were resuspended in 1 mL of sterilized water, diluted appropriately, and then applied to a YPD agar medium.
From the haploid yeast group obtained by this mutagenesis method, yeast having less mannan and immunopotentiating ability was screened by the following primary to tertiary screening.

-Primary screening of haploid-
As the primary screening, cell wall mutants were selected. The cell wall mutants were selected by using the following three types of agar medium, applying bacterial cells to each medium, culturing at 30 ° C. for 3 days, and performing the following evaluation method.

--- Medium-
The media used for the selection of the cell wall mutant strains are the following three types of media.
(1) UPD-containing YPD agar medium (1% by weight yeast extract, 2% by weight peptone, 2% by weight glucose, 6 × 10 ⁻³ % by weight uracil, 2% by weight agar)
(2) Uracil-containing 1/5 YPD agar medium (0.2% by weight yeast extract, 0.4% by weight peptone, 0.4% by weight glucose, 1.2 × 10 ⁻³ % by weight uracil, 2% by weight agar)
(3) Uracil-containing 1/5 YPD + 0.05 mass% SDS agar medium

-Evaluation method-
As a method for evaluating cell wall mutants, strains satisfying the following (1) and (2) were selected.
(1) When the uracil-containing YPD agar medium and the uracil-containing 1/5 YPD agar medium were compared, the growth was slow in the uracil-containing 1/5 YPD agar medium, and
(2) When comparing a uracil-containing 1/5 YPD agar medium with a uracil-containing 1/5 YPD + 0.05 mass% SDS agar medium, a strain that grows slowly on a uracil-containing 1/5 YPD + 0.05 mass% SDS agar medium (or Strains that die).

--Results--
As a result of the primary screening, mutants of 3,120 strains were obtained.

-Secondary screening of haploid-
--Method--
As a secondary screening, mannan staining with FITC-labeled ConA was performed. The colony was scraped off with a toothpick and suspended in PBS + 1.0 M sorbitol solution. The mixture was collected by centrifugation, resuspended in PBS + 1.0 M sorbitol solution, mixed with FITC-labeled ConA solution (manufactured by SIGMA), and allowed to stand at room temperature for 30 minutes in the dark. After washing once, the cells were observed under a blue excitation light using a fluorescence microscope. A strain was selected in which almost no fluorescence was observed when observed as a whole and fluorescence could not be confirmed on the photograph taken.

--Results--
As a result of the secondary screening, 4 strains (mutants 1 to 4) were selected from 3,120 strains obtained in the primary screening.
FIG. 1 is a microscope image of mutants 1 to 4, the upper row is an optical microscope image, and the lower row is a fluorescence microscope image observed after mannan staining. In FIG. 1, the mutants 1 to 4 have almost no fluorescence, and especially the mutants 2 to 4 have no fluorescence.

Incidentally, variant 1 is Saccharomyces cerevisiae FERM P-21313, variant 2 is Saccharomyces cerevisiae FERM P-21314, mutant 3 is a Saccharomyces cerevisiae FERM P-21354, mutant 4, Saccharomyces cerevisiae FERM P-21355, all four of which are deposited at the National Institute of Advanced Industrial Science and Technology and the Patent Biological Depositary Center.

-Tertiary screening of haploids-
--Method--
As a third screening, the macrophage activation ability of mutants 1 to 4 was measured. Mutants 1 to 4 were collected by centrifugation, treated with ethanol, and washed twice with sterilized water. Furthermore, after washing once with RPMI-1640 medium, it was resuspended in RPMI-1640 medium, and the number of yeasts was counted with a hemocytometer. A sample was prepared so that the yeast concentration was a prescribed amount, and used as a sample.
As macrophages, cells on the second day of culture were used. The cultured macrophages were collected from the culture flask, washed twice with fresh medium, and the number of cells was counted with a hemocytometer. A macrophage cell concentration was prepared so as to be a prescribed amount and used as a macrophage culture solution for activity measurement.

The macrophage culture solution was dispensed into a 24-well plate, and the sample was mixed so that the ratio of yeast to macrophage cell number was 30: 1. This was cultured at 37 ° C. and 5% CO ₂ for 6 hours, and then the culture solution was taken in a tube and centrifuged. The supernatant was recovered, and the amount of TNF-α protein contained in the supernatant was measured using Quantikine Mouse TNF-α Immunoassay (manufactured by R & D Systems). As a positive control for macrophage activation, Lipopolysaccharide (LPS, manufactured by Wako Pure Chemical Industries, Ltd.) was used.

--Results--
FIG. 2 is a diagram showing the results of a macrophage activation ability test for mutants 1 to 4. In FIG. 2, it was shown that the mutants 3 and 4 have very strong macrophage activation ability (immunostimulatory ability) compared with the parent strain (T21-U strain).
As a result of the tertiary screening, one strain (mutant 4) was selected from the four strains of mutants 1 to 4, and was used for production of glucan-exposed yeast diploids.

<Manufacture of diploid strains>
The mutant 4 was crossed with T-19 (produced by Oriental Yeast Co., Ltd.), which is a haploid of practical baker's yeast, to produce a diploid mutant 5. The mutant 5 was spore-separated and the spore-separated yeast group (hereinafter sometimes referred to as “mutant 5-spore isolate”) was used for the following primary screening.

-Primary screening of diploid-
As primary screening, uracil non-requirement and cell wall mutants were selected.

-Selection of non-uracil-requiring strains-
--- Method ---
The uracil non-requiring strain is selected using an SD medium (0.67% by mass Yeast nitrogen base without amino acids (Difco), 2% by mass glucose, 2% by mass agar), and spotting the cells on the SD medium. The strains that grew were selected.

-Selection of cell wall mutants-
Selection of the cell wall mutant strain was carried out by the following evaluation method after spotting microbial cells on each medium and culturing at 30 ° C. for 3 days.

--- Medium ---
The following three types of media were used as the media.
(1) Uracil-containing 1/5 YPD agar medium (0.2 mass% yeast extract, 0.4 mass% peptone, 0.4 mass% glucose, 6 × 10 ⁻³ mass% uracil, 2 mass% agar)
(2) Uracil-containing 1/5 YPD + 0.02 mass% SDS agar medium (3) Uracil-containing 1/5 YPD + 0.05 mass% SDS agar medium

--- Evaluation method ---
As a method for evaluating cell wall mutants, strains satisfying the following (1) and (2) were selected.
(1) When comparing a uracil-containing 1/5 YPD agar medium and a uracil-containing 1/5 YPD + 0.02 mass% SDS agar medium, the growth of the uracil-containing 1/5 YPD + 0.02 mass% SDS agar medium is slow, and
(2) When comparing uracil-containing 1/5 YPD + 0.02 mass% SDS agar medium with uracil-containing 1/5 YPD + 0.05 mass% SDS agar medium, it grows on uracil-containing 1/5 YPD + 0.05 mass% SDS agar medium. Slow strains (or strains that die).

-Mating matrix judgment-
The mating type (α type strain or a type strain) of the haploid strain (mutant 5 spore isolate) obtained by the primary screening was confirmed by the following method, and mating matrix determination was performed.

--Confirmation of joint type--
The YPD medium was dispensed into a 96-well plate, and the mutant 5 segregant was inoculated with a toothpick, followed by stationary culture at 30 ° C. overnight. Inoculate a tester strain (a strain of which junction type is known) X-2180A (manufactured by ATCC) and X-2180B (manufactured by ATCC) into a φ16 mm test tube containing YPD medium, and shake overnight at 30 ° C. Cultured. YPD medium was dispensed into a 24-well plate, and a segregant culture solution was added. Furthermore, the culture solution of X-2180A or X-2180B was added, and static culture was performed at 30 ° C. for 4 hours. Then, the joining type | mold was confirmed by observing aggregation visually.

-Creation of haploid strain-
The α-type strain and the a-type strain were each grown in a YPD medium by culturing at 30 ° C. for 1 day. Both the numbers of bacteria were set to the same level, and both yeasts were placed in a new YPD medium and cultured at 30 ° C. for 12 hours. The zygote was isolated, spread on a YPD agar medium, and cultured with shaking at 30 ° C. for 1 to 2 days to isolate a relatively large colony as a diploid. About the isolate | separated colony, it was observed that it did not have joining property, and became larger than the haploid under the microscope, and the formation of the diploid was confirmed. 70 strains of diploid were obtained.
In addition, since these strains were able to grow and proliferate in a YPD medium, it was recognized that they have osmotic pressure resistance despite a small amount of mannan.

-Secondary screening of diploid-
--Method--
As a secondary screening, mannan staining with FITC-labeled ConA was performed. Mannan staining was performed in the same manner as the secondary screening of the haploid. The secondary screening was performed 3 times to confirm reproducibility.

--Results--
As a result of the secondary screening, 3 strains (mutants 6 to 8) were selected from the 70 diploid strains.
FIG. 3 is microscopic images of mutants 6 to 8, in which the upper part is an optical microscopic image and the lower part is a fluorescent microscopic image observed after mannan staining.

Mutant 6 is Saccharomyces cerevisiae FERM P-21728, Mutant 7 is Saccharomyces cerevisiae FERM P-21729, Mutant 8 is Saccharomyces cerevisiae FERMia 17 FERM It is deposited at the National Institute of Advanced Industrial Science and Technology and the Patent Biological Depositary Center.

-Macrophage activation ability-
Macrophage activation ability of the mutants 6-8 was measured by the following method.

-Sample processing-
Mutants 6-8 in the logarithmic growth phase (12 hours of culture) were collected by centrifugation, treated with 70% by mass ethanol for 60 minutes, and then washed twice with sterilized water. Furthermore, after washing once with RPMI-1640 medium (manufactured by SIGMA), it was resuspended in RPMI-1640 medium and the number of yeasts was counted with a hemocytometer. A sample was prepared so that the yeast concentration would be 3.0 × 10 ⁷ cells / mL.

--- Preparation of cells-
As mouse macrophage cells (RAW264.7, manufactured by ATCC), cells on the second day of culture were used. The cultured RAW264.7 cells were collected from the culture flask, washed twice with fresh RPMI-1640 medium, and then counted with a hemocytometer. The RAW264.7 cell concentration was adjusted to 1.0 × 10 ⁶ cells / mL, and used as a RAW264.7 cell culture solution for activity measurement.

--Measurement of macrophage activation ability--
0.25 mL of RAW264.7 cell culture solution was dispensed into a 24-well plate, and the sample was mixed so that the ratio of the number of cells of yeast to macrophage was 30: 1. This was cultured at 37 ° C. under 5% CO ₂ for 6 hours, and then the culture solution was taken in a tube and centrifuged at 2,000 × g for 1 minute. The supernatant was collected and the amount of TNF-α protein contained in the supernatant was measured using Quantikine (registered trademark) Mouse TNF-α Immunoassay (R & D systems). For the measurement, MICROPLATE READER Model-680 (manufactured by BIO-RAD) was used. As positive control for macrophage activation, Lipopolysaccharide (LPS, manufactured by Wako Pure Chemical Industries, Ltd.) 0.2 μg / mL was used.

--Results--
FIG. 4 is a diagram showing the results of a macrophage activation ability test for mutants 6-8. In FIG. 4, the wild type diploid strains are T-19 strain (produced by Oriental Yeast Co., Ltd.) and T-21 strain (produced by Oriental Yeast Co., Ltd.) which are haploids of practical bread yeast. This is a crossed strain.
The TNF-α concentration of the wild-type diploid strain was about 900 pg / mL, whereas mutant 6 was about 1,750 pg / mL, mutant 7 was about 2,250 pg / mL, and mutant 8 was A high activity of 2,100 pg / mL was confirmed.
From these results, it was shown that the mutants 6 to 8 had a higher TNF-α production inducing ability and a very strong macrophage activation ability (immunostimulatory ability) than the wild type diploid.

(Example 2: Comparison of macrophage activation ability with practical strains)
The macrophage activating ability of the mutant 6, the mutant 7, and the mutant 8, and the macrophage activating ability of practical strains A, B, C, and D (all manufactured by Oriental Yeast Co., Ltd.) are as follows: Measured by the method and compared.
The number of genomes of the practical strains A to D is diploid yeast.

<Sample processing>
One platinum ear of each strain was inoculated into 5 mL of YPD medium (1% by mass yeast extract, 2% by mass peptone, 2% by mass glucose), and cultured with shaking at 30 ° C. for 2 days. The yeast suspension obtained by the culture was washed twice with distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.), and then the dry (DM) mass of each strain was measured. The cells were collected by centrifugation, treated with 70% by mass ethanol for 60 minutes, washed twice with RPMI-1640 medium, and then 1,000 μg (DM mass) in RPMI-1640 medium containing 10% by mass bovine serum (manufactured by CELLECT). ) / ML.

<Preparation of cells>
RAW264.7 was cultured in the same manner as in Example 1, and a cell suspension was prepared to 1.0 × 10 ⁶ cells / mL.

<Measurement of macrophage activation ability>
RAW264.7 cell culture solution was dispensed into a 48-well plate at 0.5 mL, and cultured for 2 hours under conditions of 37 ° C., humidity 100%, and 5% CO ₂ . After confirming adhesion of RAW264.7 cells, the culture supernatant was removed, 0.5 mL of the sample 1,000 μg / mL was added, and the cells were cultured for 24 hours. The culture supernatant was collected, and the amount of TNF-α protein was measured in the same manner as in Example 1. Lipopolysaccharide (LPS, manufactured by Wako Pure Chemical Industries, Ltd.) 0.001 μg / mL was used as a positive control for macrophage activation.

<Result>
FIG. 5: A is a figure which shows the result of the macrophage activation ability test about utility strain AD, and the variants 6-8. FIG. 5B is a graph showing the activation ability of LPS-stimulated macrophages used as positive controls.
The TNF-α concentration of the practical strain was about 300 pg / mL to 500 pg / mL, and the TNF-α concentration of the wild type diploid strain was about the same as about 600 pg / mL.
On the other hand, the mutant 6 was about 800 pg / mL, the mutant 7 was about 1,000 pg / mL, and the mutant 8 was about 800 pg / mL, both of which were confirmed to have higher activities than the wild type diploid strain. . Among these, Mutant 7 showed the highest activity.
From these results, the mutants 6 to 8 have a higher ability to induce TNF-α production and have a very strong macrophage activation ability (immunostimulatory ability) compared to wild-type diploid strains and practical strains. It has been shown.

(Example 3: Manufacturability)
About the mutants 6-8, examination of manufacturing suitability was performed by the following method. As a comparative control, haploid mutant 4 was used.

<Sugar yield>
-Method-
Mutant 4 and mutants 6-8 cultured in a uracil-containing YPD agar medium (composition is the same as in Example 1) were each inoculated with 1 platinum ear in 5 mL of 6 × 10 ⁻³ mass% uracil YPD liquid medium. The shaking culture was performed overnight at 30 ° C. Each mutant suspension obtained in the culture was washed with distilled water, and the dry (DM) mass of each strain was measured.
The sugar yield was calculated from the wet mass (g) of each mutant recovered per 1 g of sugar, assuming that the water content of yeast was 67% by mass.
The results are shown in Table 1 below.

-Result-
Variants 6-8, which are diploid strains, showed a sugar yield of 80% by mass or more, whereas mutant 4, which is a haploid strain, had an extremely high yield of sugar, 4% by mass. (Table 1).
From these results, the growth ability of the mutant 4 which is a haploid strain is low, and the mutants 6 to 8 which are diploid strains have a very high growth ability compared to the mutant 4. Admitted.

<Nutrition requirement>
-Method-
An agar medium (hereinafter sometimes referred to as “uracil-added medium”) to which 2 × 10 ⁻³ mass% uracil is added to SD medium, or an agar medium to which uracil is not added (hereinafter referred to as “medium without uracil”). The mutant 4 and the mutants 6 to 8 were applied to each other, cultured at 30 ° C. for 2 days, and the degree of growth was confirmed.

-Result-
FIG. 6A shows the result of the medium added with uracil, and FIG. 6B shows the result of the medium not added with uracil.
From these results, it was confirmed that mutant 4 was uracil-requiring because it grew in the uracil-added medium but did not grow in the uracil-free medium.
On the other hand, since it was confirmed that the mutants 6 to 8 grew both in the uracil-added medium and in the uracil-free medium, the mutants 6 to 8 are non-uracil-requiring strains and are low in production. It was found that it could be manufactured at cost.

<Cohesiveness>
-Method-
Mutant 4 and Mutant 6-8 were each inoculated with 1 platinum ear in 5 mL of 6 × 10 ⁻³ mass% uracil YPD liquid medium, and cultured with shaking at 30 ° C. overnight. The cell morphology of each mutant after culture was observed at 400 times with an optical microscope.

-Result-
A microscopic image of each mutant is shown in FIG.
Variant 4, which is a haploid strain, had very high aggregation properties. Mutant 8 was also found to aggregate, but the degree of aggregation was lower than that of mutant 4. Mutant 4 caused clogging of the separator and had a manufacturing problem, while mutant 8 did not cause clogging of the separator and had no manufacturing problem. Mutants 6-7 were not found to be cohesive.
From these results, it was confirmed that the diploid strain is superior in production suitability because it has lower aggregation than the haploid strain.

(Example 4: Production of bread)
Mutants 6-8 were used to make bread. As a comparative control, a wild type diploid strain was used.

<Manufacture of pressed yeast>
Fed-batch culture was carried out using mutants 6 to 8, cultured strain E, and wild-type diploid strain cultured in YPD liquid medium as seed yeast. Under conditions of 1,400 g of sugar (converted to sucrose), 140 g of urea, and 28 g of monosodium phosphate, the mixture was continuously fed with a volume of 15 L using a 30 L jar fermenter and cultured. The fed-batch method followed a conventional method. After completion of the culture, the culture broth was separated, washed with water, and filtered to obtain a compressed live yeast.

<Method of manufacturing bread>
The bread was produced with reference to the Japan East Industry Association (August 1996). That is, sugar and salt are dissolved using a part of water and added to Million (Nisshin Flour Milling Co., Ltd.) and Number One (Nisshin Flour Milling Co., Ltd.) in the mixer bowl, and the rest Of water was added. Subsequently, the mutant yeast 6-8 produced as described above, the practical strain E, and the compressed yeast of any of the wild-type diploid strains and shortening (manufactured by Miyoshi Oil & Fats Co., Ltd.) are added to the mixer bowl and mixed. Went. Mixing is performed at 28 ° C. with a bread mixer (manufactured by Kanto Blender Kogyo Co., Ltd.) at low speed (L) for 1 minute and medium speed (M) for 4 minutes. 10 times. After kneading, the bread dough was transferred to a beaker, and the time until the bread dough became 1,750 mL was measured in a 28 ° C. incubator, and the first fermentation was performed. After degassing by punching 5 to 6 times, the mixture was returned to the beaker and further subjected to second fermentation at 28 ° C. for 30 minutes, and the volume of bread dough was measured from the scale of the beaker. After fermentation, it was divided into 360 g, rounded and then benched for 20 minutes at 28 ° C. Next, a molder (standard loaf mold) was molded, and the time until it reached 2 cm on the mold was measured in a proofer at 38 ° C. and a relative humidity of 85%. After the proof fermentation, it was baked at 200 ° C. for 20 minutes. The bread volume (cm ² ), length (mm), width (mm), height (mm), and mass (g) of the bread after baking were measured.
Table 2 shows the composition of each material.

<Result>
Table 3 shows the results of measuring the time of the first fermentation, the fermentation time of the proof, the volume, length, width, height, and mass of the bread after baking. Moreover, the bread after baking is shown in FIG.

From Table 3 and FIG. 8, the breads obtained from the mutants 6 to 8 are practical strains E, in the first fermentation time, proof fermentation time, volume, length, width, height, mass, and external shape. It was completely different from the wild type diploid strain. In addition, when the breads obtained from the mutants 6 to 8 were eaten, the flavors such as aroma and taste and the texture were not different from those of the practical strain E and the wild type diploid strain. It was.

(Example 5: Production of dry yeast powder)
Mutants 6-8 and wild type diploid strains were used to produce dry yeast powder.
Mutant 6-8 and the wild type diploid strain were each suspended in water, and this suspension was spray-dried to a loss on drying of less than 7% by mass with a spray dryer (Okawara Chemical Co., Ltd.). . At this time, the hot air temperature was 185 ° C., the exhaust temperature was 100 ° C., and the treatment amount was 3 kg / hour.
When the dried yeast powder obtained from the mutants 6 to 8 was eaten, the flavor such as aroma and taste and the texture were completely the same as the dried yeast powder of the wild type diploid strain.

(Example 6: Production of scones)
Mutants 6-8 and wild type diploid strains were used to produce scones.
Add 4g of mutant 6-8, wild type diploid strain, and baking powder, sugar, salt, fresh cream, oil, milk, etc. as appropriate to 100g of soft flour, mix at 24 ° C and knead. After raising, proofing was performed for 30 minutes at 30 ° C. and 75% relative humidity, followed by firing at 200 ° C. A well-fermented top-standing scone was produced.
When the scones obtained from the mutants 6 to 8 were eaten, the flavors such as aroma and taste and the texture were completely the same as those of the wild type diploid strain.

(Example 7: Production of dog food)
Mutants 6-8 were used to produce dog food.
Add any of mutants 6 to 8 so that the dry content in the solid pellet is 3% by mass in the feed material of the animal experiment feed DS-A (made by Oriental Yeast Co., Ltd.) for dogs, Stirring, adding water, kneading, molding into pellets, and drying. It could be produced in the same way as the normal product DS-A.

FERM P-21313
FERM P-21314
FERM P-21354
FERM P-21355
FERM P-21728
FERM P-21729
FERM P-21730

  Since the yeast of the present invention has an excellent immunostimulatory ability and can be safely ingested, it improves the immune function of consumers whose immune function has been reduced, as well as for diseases such as cancer, allergic symptoms, and infectious diseases. It can be suitably used for prevention and treatment. Specifically, the yeast can be used for food production such as use for food materials, feeds, feeds, etc., and fermentation of bread and beer.

Claims (2)

Less mannan cell walls have immunopotentiating capability, and can be grown in YPD liquid medium osmolality 300 meters Osm is the yield based on sugar is 75 mass% or more state, and are the number of genome diploid or ,
Saccharomyces cerevisiae FERM P-21728, Saccharomyces cerevisiae FERM P-21729, and Saccharomyces cerevisiae FERM any der Yeast characterized by Rukoto of P-21 730. A food or feed containing the yeast according to claim 1.