WO2024150575A1 - Feedstuff for animals, food product material, and method for extracting yeast cell walls - Google Patents

Feedstuff for animals, food product material, and method for extracting yeast cell walls Download PDF

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WO2024150575A1
WO2024150575A1 PCT/JP2023/044211 JP2023044211W WO2024150575A1 WO 2024150575 A1 WO2024150575 A1 WO 2024150575A1 JP 2023044211 W JP2023044211 W JP 2023044211W WO 2024150575 A1 WO2024150575 A1 WO 2024150575A1
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yeast cell
yeast
ypd
cell wall
cell walls
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PCT/JP2023/044211
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French (fr)
Japanese (ja)
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善則 村田
喜永 大谷
有宏 岩下
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国立研究開発法人国際農林水産業研究センター
明治飼糧株式会社
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Publication of WO2024150575A1 publication Critical patent/WO2024150575A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/35—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from potatoes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A23L31/10—Yeasts or derivatives thereof
    • A23L31/15—Extracts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage

Definitions

  • the present invention relates to animal feed, food ingredients, and a method for extracting yeast cell walls.
  • Patent Document 1 describes a method for producing animal or fish feed in which cassava residue is fermented with starch-degrading enzymes and microorganisms.
  • Patent Document 1 The method for producing animal or fish feed described in Patent Document 1 makes it possible to obtain animal feed using cassava residues and other materials that have not previously had any use. However, Patent Document 1 does not provide any information about the active ingredients in this animal feed.
  • the present invention aims to provide animal feed, food materials, and a method for extracting yeast cell walls that can regulate the proliferation of immune cells or the immune system.
  • an animal feed comprising yeast cell walls extracted from yeast cultured using potato residue, which is the residue after extracting starch from potatoes, When the contents of ⁇ -glucan and chitin in the yeast cell wall are measured, both of the conditions shown in the following formula (F1) and the following formula (F2) are satisfied. Animal feed.
  • the value of G YPD /G is 1.19 or more;
  • the C YPD /C value is 0.98 or less.
  • the tuber is at least one selected from the group consisting of potato, sweet potato, Jerusalem artichoke, apios, taro, taro, konjac, yam, cassava, Chinese yam, and Chinese yam; Animal feed.
  • the animal feed according to any one of [1] to [3], The animal is at least one selected from the group consisting of dairy cattle, beef cattle, sheep, pigs, chickens, and fish; Animal feed.
  • a food material comprising a yeast cell wall extracted from a yeast cultured using potato residue, which is the residue remaining after extracting starch from potatoes, When the contents of ⁇ -glucan and chitin in the yeast cell wall are measured, both of the conditions shown in the following formula (F1) and the following formula (F2) are satisfied. Food ingredients.
  • YPD ⁇ -glucan content (mass%) in yeast cell walls extracted from yeast cultured using YPD medium
  • the method includes a step of extracting a yeast cell wall that satisfies the following condition 1 from the yeast.
  • 1 is a graph showing the ⁇ -glucan content in yeast cell walls (CP and YPD) obtained in Example 1 and Comparative Example 1.
  • 1 is a graph showing the ⁇ -glucan content in yeast cell walls (SP and YPD) obtained in Example 2 and Comparative Example 2.
  • 1 is a graph showing the chitin content in yeast cell walls (CP and YPD) obtained in Example 1 and Comparative Example 1.
  • 1 is a graph showing the chitin content in yeast cell walls (SP and YPD) obtained in Example 2 and Comparative Example 2. This is a graph showing the results of cell proliferation when peripheral blood mononuclear cells (PBMCs) were grown using the yeast cell wall (P.
  • PBMCs peripheral blood mononuclear cells
  • Kudriavzevii obtained in Example 1 and reagents (Concanavalin A and Zymosan).
  • 1 is a graph showing the results of evaluating the effects on cytokines (tumor necrosis factor, TNFa) when the yeast cell wall (P. Kudriavzevii) obtained in Example 1 and a yeast cell wall reagent (Zymosan) are used.
  • 1 is a graph showing the results of evaluating the effects on cytokines (interleukin-1 ⁇ , IL-1b) when the yeast cell wall (P. Kudriavzevii) obtained in Example 1 and a yeast cell wall reagent (Zymosan) are used.
  • the animal feed according to the present embodiment is an animal feed made of yeast cell walls extracted from yeast cultured using potato residue, which is the residue remaining after extracting starch from potatoes.
  • yeast cell walls extracted from yeast cultured using potato residue, which is the residue remaining after extracting starch from potatoes.
  • G YPD /G is preferably 1.05 or more, more preferably 1.1 or more, and particularly preferably 1.19 or more.
  • the upper limit of the value of G YPD /G is not particularly limited.
  • the value of G YPD /G may be 10 or less, or 5 or less.
  • C YPD /C is preferably 0.98 or less, more preferably 0.94 or less.
  • the lower limit of C YPD /C is not particularly limited.
  • the value of C YPD /C may be 0.1 or more, or 0.2 or more.
  • the animal feed of this embodiment is preferably used for at least one animal selected from the group consisting of dairy cows, beef cattle, sheep, pigs, chickens, and fish.
  • the potato residue used in this embodiment is the residue remaining after starch is extracted from potatoes.
  • tubers include potato, sweet potato, Jerusalem artichoke, apios, taro, taro, konjac, yam, cassava, Chinese yam, and Chinese yam.
  • sweet potato or cassava is preferred from the viewpoint of raw material availability.
  • yeast used in this embodiment is not particularly limited.
  • yeast include Pichia kudriavzevii, Saccharomyces cerevisiae, and Kluyveromyces marxianus.
  • the yeast cell wall extraction method according to the present embodiment includes a step of ethanol fermenting potato residue, which is the residue after extracting starch from potatoes, with starch-degrading enzymes and yeast while culturing the yeast to obtain an ethanol fermented product (hereinafter also referred to as the "first step"), a step of separating the yeast from the ethanol fermented product (hereinafter also referred to as the "second step"), and a step of extracting yeast cell walls from the yeast that satisfy the following condition 1 (hereinafter also referred to as the "third step”).
  • the conditions shown in both the formula (F1) and the formula (F2) are satisfied.
  • the yeast cell wall extraction method according to this embodiment allows the animal feed according to the embodiment described above to be produced efficiently.
  • potato residue which is the residue remaining after extracting starch from potatoes.
  • potatoes they are as mentioned above.
  • the water content of the potato residue is not particularly limited, however, from the viewpoint of saccharification and fermentation, the water content of the potato residue is preferably 30% by mass or more.
  • dried potato residue may be used. When dried potato residue is used, water may be added to adjust the moisture content of the potato residue. Also, the dried potato residue may be mixed with the potato residue having a high moisture content after starch extraction.
  • the potato residue is then subjected to ethanol fermentation using starch-degrading enzymes and yeast, while the yeast is cultured, to obtain an ethanol fermented product.
  • the potato residue may be fermented as is, but it is preferable to heat treat it before fermentation in order to gelatinize the starch remaining in the potato residue.
  • heating conditions a heating temperature of 60°C or higher and a heating time of 5 minutes or longer are preferable.
  • a heating temperature of 60°C or higher can increase the gelatinization efficiency of the starch remaining in the potato residue.
  • the heating time is not particularly limited and can be set appropriately taking into account the heating temperature.
  • the treatment with the starch-degrading enzyme and the fermentation treatment with yeast can be carried out separately or simultaneously.
  • the yeast is as described above.
  • the starch-degrading enzyme is not particularly limited, and may be any enzyme that degrades starch. Examples of the starch-degrading enzyme include ⁇ -amylase and glucoamylase. In order to reduce the viscosity of the potato residue, pectinase or cellulase may be used in combination with the starch-degrading enzyme.
  • the amount of ⁇ -amylase added is preferably 9 ⁇ 10 ⁇ 5 units (hereinafter also referred to as “U”) to 600 U per gram of potato residue solid content, and more preferably 8 ⁇ 10 ⁇ 3 U to 20 U.
  • the amount of glucoamylase added is preferably 3 ⁇ 10 ⁇ 4 U to 200 U per gram of potato residue solid content, and more preferably 3 ⁇ 10 ⁇ 2 U to 0.2 U.
  • the amount added is preferably 1 ⁇ 10 ⁇ 4 U to 100 U per gram of potato residue solid content.
  • pectinase the amount added is preferably 1 ⁇ 10 ⁇ 3 U to 1000 U, more preferably 1 ⁇ 10 ⁇ 1 U to 1 U, per gram of potato residue solid content.
  • the yeast is separated from the ethanol fermentate.
  • Liquid components such as ethanol contained in the ethanol fermentation product can be removed by at least one of a method of squeezing the ethanol fermentation product and a method of heating the ethanol fermentation product. By removing the liquid components from the ethanol fermentation product, the yeast can be separated.
  • the yeast cell walls are extracted from the yeast.
  • the pulverization method may be dry pulverization or wet pulverization.
  • Examples of the pulverization apparatus include a bead mill, a ball mill, and a colloid mill.
  • the resulting pulverized product is washed with ethanol or the like, and then the solid content is precipitated to fractionate the yeast cell walls, allowing the yeast cell walls to be extracted. In this manner, yeast cell walls that satisfy condition 1 above can be extracted.
  • the food material according to the present embodiment is the same as the animal feed according to the present embodiment described above.
  • the animal feed according to this embodiment can regulate the proliferation of immune cells or the immune system. Therefore, as a food ingredient, it is expected to have effects such as health promotion.
  • Example 1 20 g of dried cassava residue was mixed with 80 g of pure water to obtain a cassava residue with a moisture content of 80% by mass. 8 to 10 units of ⁇ -amylase per 1 g of cassava residue solid content was added to the obtained cassava residue, and the mixture was heat-treated at a temperature of 30 to 100° C. for 0.4 to 1 hour, and then cooled to obtain a medium. To the obtained medium, an enzyme cocktail (glucoamylase 1.28 units, pectinase 4.0 units, cellulase 0.46 units) and a nitrogen source (yeast extract + peptone) were added, and further, Pichia kudriavzevii was inoculated as a yeast.
  • an enzyme cocktail glucoamylase 1.28 units, pectinase 4.0 units, cellulase 0.46 units
  • a nitrogen source yeast extract + peptone
  • the yeast was cultured under conditions of a temperature of 30 to 40°C, an agitation speed of 50 to 150 rpm, and for 24 to 40 hours to obtain an ethanol fermentation product.
  • the obtained ethanol fermentation product was squeezed to remove the liquid containing ethanol, and the yeast was separated.
  • the separated yeast was then autolyzed and crushed using a bead mill, washed with ethanol, and the solids were then precipitated to fractionate and extract the yeast cell walls.
  • Example 2 The medium and yeast cell walls were obtained in the same manner as in Example 1, except that 20 g of dried sweet potato residue was used instead of 20 g of dried cassava residue.
  • Example 1 Yeast cell walls were obtained in the same manner as in Example 1, except that YPD medium was used as the medium.
  • Example 2 Yeast cell walls were obtained in the same manner as in Example 2, except that YPD medium was used as the medium.
  • Example 1 and Comparative Example 1 The ⁇ -glucan contents in the yeast cell walls obtained in Example 1 and Comparative Example 1 are shown in Figure 1.
  • the ⁇ -glucan contents in the yeast cell walls obtained in Example 2 and Comparative Example 2 are shown in Figure 2.
  • the chitin contents in the yeast cell walls obtained in Example 1 and Comparative Example 1 are shown in Figure 3.
  • the chitin contents in the yeast cell walls obtained in Example 2 and Comparative Example 2 are shown in Figure 4. 1 to 4
  • Example 1 uses a medium made of cassava residue as a raw material, and is therefore indicated as CP
  • Example 2 uses a medium made of sweet potato residue as a raw material, and is therefore indicated as SP.
  • Comparative Examples 1 and 2 use a YPD medium, and are therefore indicated as YPD.
  • the results are indicated by error bars in Figures 1, 2, and 4.
  • the results of the first test are shown on the left side
  • the results of the second test are shown on the right side.
  • Example 1 had a G YPD /G value of more than 1 and a C YPD /C value of less than 1.
  • Example 2 had a G YPD /G value of more than 1 and a C YPD /C value of less than 1.
  • PBMC peripheral blood mononuclear cells
  • Example 1 uses Pichia kudriavzevii as yeast, and is therefore indicated as P. kudriavzevii, and the example in which no yeast cell wall or reagent was added to the sample is indicated as Control, and the rest are indicated by the abbreviations of the reagent names.
  • the abbreviations are as follows: ConA: Concanavalin A (3 ⁇ g/mL) Zymosan: ⁇ -glucan standard (100ug/mL) CP: Cassava residue From the results shown in Figure 5, it was confirmed that the yeast cell wall obtained in Example 1 has a significantly higher PBMC proliferation effect than Zymosan, a yeast cell wall reagent. It was also confirmed that the yeast cell wall obtained in Example 1 has a higher PBMC proliferation effect than Concanavalin A, an immune cell activation reagent.
  • the yeast cell wall obtained in Example 1 and Zymosan were evaluated for their effects on cytokines as follows.
  • As cytokines tumor necrosis factor (TNF ⁇ ) and interleukin-1 ⁇ (IL-1b) were evaluated. That is, yeast cell walls and Zymosan were added to PBMCs isolated from bovine blood, and the PBMCs were stimulated at 37°C for 6 hours. RNA was then extracted, and the gene expression level of each cytokine relative to the standard gene ⁇ -Actin was measured by quantitative PCR to evaluate the effect of the yeast cell walls on cytokine production.
  • TNF ⁇ tumor necrosis factor
  • IL-1b interleukin-1 ⁇
  • FIG. 6 shows the results of evaluating the effects of the yeast cell wall obtained in Example 1 and the yeast cell wall reagent on tumor necrosis factor (TNF ⁇ ).
  • FIG. 7 shows the results of evaluating the effect on interleukin-1 ⁇ (IL-1b) when the yeast cell wall obtained in Example 1 and a yeast cell wall reagent are used.
  • Example 1 uses Pichia kudriavzevii as yeast, and is therefore indicated as P. kudriavzevii, and Zymosan is the name of the reagent.
  • the example in which the yeast cell wall was not used is indicated as Control.

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Abstract

This feedstuff for animals is yeast cell walls extracted from yeast cultured using potato residues which are residues obtained from potatoes from which starch has been taken out. When the β-glucan content and chitin content in the yeast cell walls are measured, it is found that the feedstuff for animals satisfies both conditions represented by formula (F1) and formula (F2). (F1): GYPD / G > 1; (F2): CYPD / C < 1, where: G is the β-glucan content (mass%) in the yeast cell walls; C is the chitin content (μmol/mg) in the yeast cell walls; GYPD is the β-glucan content (mass%) in the yeast cell walls extracted from yeast cultured using a YPD medium; and CYPD is the chitin content (μmol/mg) in the yeast cell walls extracted from yeast cultured using a YPD medium.

Description

動物用飼料、食品素材、および酵母細胞壁の抽出方法Animal feed, food material, and method for extracting yeast cell wall
　本発明は、動物用飼料、食品素材、および酵母細胞壁の抽出方法に関する。 The present invention relates to animal feed, food ingredients, and a method for extracting yeast cell walls.
　芋類からでんぷんを取り出した後の残渣の利用方法として、動物用飼料などが検討されている。例えば、特許文献１には、キャッサバ残渣をでんぷん分解酵素と微生物とで発酵処理する、動物または魚類用飼料の製造方法が記載されている。 Animal feed and other uses for the residue left over after extracting starch from tubers are being considered. For example, Patent Document 1 describes a method for producing animal or fish feed in which cassava residue is fermented with starch-degrading enzymes and microorganisms.
特開２０１７－０３５００５号公報JP 2017-035005 A
　特許文献１に記載の動物または魚類用飼料の製造方法によれば、従来は用途のなかったキャッサバ残渣などを利用した動物用飼料を得られる。しかしながら、特許文献１においては、この動物用飼料中の有効成分についての知見はなかった。 The method for producing animal or fish feed described in Patent Document 1 makes it possible to obtain animal feed using cassava residues and other materials that have not previously had any use. However, Patent Document 1 does not provide any information about the active ingredients in this animal feed.
　本発明は、免疫細胞の増殖、または、免疫を調節できる動物用飼料、食品素材、および酵母細胞壁の抽出方法を提供することを目的とする。 The present invention aims to provide animal feed, food materials, and a method for extracting yeast cell walls that can regulate the proliferation of immune cells or the immune system.
　本発明の一態様によれば、以下の動物用飼料、食品素材、および酵母細胞壁の抽出方法が提供される。
［１］　芋類からでんぷんを取り出した後の残渣である芋残渣を用いて培養した酵母から抽出した酵母細胞壁である動物用飼料であって、
　前記酵母細胞壁中のβ－グルカンおよびキチンの含量を測定した場合に、下記数式（Ｆ１）および下記数式（Ｆ２）で示す両方の条件を満たす、
　動物用飼料。
ＧＹＰＤ／Ｇ　＞　１　・・・（Ｆ１）
ＣＹＰＤ／Ｃ　＜　１　・・・（Ｆ２）
Ｇ：前記酵母細胞壁中のβ－グルカンの含量（質量％）
Ｃ：前記酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
ＧＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のβ－グルカンの含量（質量％）
ＣＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
［２］　［１］に記載の動物用飼料において、
　前記ＧＹＰＤ／Ｇの値が、１．１９以上であり、
　前記ＣＹＰＤ／Ｃの値が、０．９８以下である、
　動物用飼料。
［３］　［１］または［２］に記載の動物用飼料において、
　前記芋類が、ジャガイモ、サツマイモ、キクイモ、アピオス、タロイモ、サトイモ、コンニャクイモ、ヤムイモ、キャッサバ、ナガイモ、およびヤマノイモからなる群から選択される少なくとも１つである、
　動物用飼料。
［４］　［１］から［３］のいずれかに記載の動物用飼料において、
　前記動物が、乳牛、肉牛、羊、豚、鶏、および魚類からなる群から選択される少なくとも１つである、
　動物用飼料。
［５］　芋類からでんぷんを取り出した後の残渣である芋残渣を用いて培養した酵母から抽出した酵母細胞壁である食品素材であって、
　前記酵母細胞壁中のβ－グルカンおよびキチンの含量を測定した場合に、下記数式（Ｆ１）および下記数式（Ｆ２）で示す両方の条件を満たす、
　食品素材。
ＧＹＰＤ／Ｇ　＞　１　・・・（Ｆ１）
ＣＹＰＤ／Ｃ　＜　１　・・・（Ｆ２）
Ｇ：前記酵母細胞壁中のβ－グルカンの含量（質量％）
Ｃ：前記酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
ＧＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のβ－グルカンの含量（質量％）
ＣＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
［６］　芋類からでんぷんを取り出した後の残渣である芋残渣を、でんぷん分解酵素と酵母とでエタノール発酵しつつ、前記酵母を培養して、エタノール発酵物を得る工程と、
　前記エタノール発酵物から前記酵母を分離する工程と、
　前記酵母から、下記条件１を満たす酵母細胞壁を抽出する工程と、を備える、
　酵母細胞壁の抽出方法。
（条件１）
　前記酵母細胞壁中のβ－グルカンおよびキチンの含量を測定した場合に、下記数式（Ｆ１）および下記数式（Ｆ２）で示す両方の条件を満たす。
ＧＹＰＤ／Ｇ　＞　１　・・・（Ｆ１）
ＣＹＰＤ／Ｃ　＜　１　・・・（Ｆ２）
Ｇ：前記酵母細胞壁中のβ－グルカンの含量（質量％）
Ｃ：前記酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
ＧＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のβ－グルカンの含量（質量％）
ＣＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ） According to one aspect of the present invention, there are provided the following animal feed, food material, and method for extracting yeast cell walls.
[1] An animal feed comprising yeast cell walls extracted from yeast cultured using potato residue, which is the residue after extracting starch from potatoes,
When the contents of β-glucan and chitin in the yeast cell wall are measured, both of the conditions shown in the following formula (F1) and the following formula (F2) are satisfied.
Animal feed.
GYPD /G>1...(F1)
CYPD /C < 1...(F2)
G: β-glucan content (mass%) in the yeast cell wall
C: chitin content in the yeast cell wall (μmol/mg)
G YPD : β-glucan content (mass%) in yeast cell walls extracted from yeast cultured using YPD medium
C YPD : chitin content (μmol/mg) in yeast cell walls extracted from yeast cultured in YPD medium
[2] In the animal feed according to [1],
the value of G YPD /G is 1.19 or more;
The C YPD /C value is 0.98 or less.
Animal feed.
[3] In the animal feed according to [1] or [2],
The tuber is at least one selected from the group consisting of potato, sweet potato, Jerusalem artichoke, apios, taro, taro, konjac, yam, cassava, Chinese yam, and Chinese yam;
Animal feed.
[4] The animal feed according to any one of [1] to [3],
The animal is at least one selected from the group consisting of dairy cattle, beef cattle, sheep, pigs, chickens, and fish;
Animal feed.
[5] A food material comprising a yeast cell wall extracted from a yeast cultured using potato residue, which is the residue remaining after extracting starch from potatoes,
When the contents of β-glucan and chitin in the yeast cell wall are measured, both of the conditions shown in the following formula (F1) and the following formula (F2) are satisfied.
Food ingredients.
GYPD /G>1...(F1)
CYPD /C < 1...(F2)
G: β-glucan content (mass%) in the yeast cell wall
C: chitin content in the yeast cell wall (μmol/mg)
G YPD : β-glucan content (mass%) in yeast cell walls extracted from yeast cultured using YPD medium
C YPD : chitin content (μmol/mg) in yeast cell walls extracted from yeast cultured in YPD medium
[6] A step of fermenting potato residue, which is the residue after removing starch from potatoes, into ethanol using a starch-degrading enzyme and yeast while culturing the yeast to obtain an ethanol fermented product;
Separating the yeast from the ethanol fermentation product;
The method includes a step of extracting a yeast cell wall that satisfies the following condition 1 from the yeast.
Methods for extracting yeast cell walls.
(Condition 1)
When the contents of β-glucan and chitin in the yeast cell wall are measured, the contents satisfy both the conditions shown in the following formula (F1) and the following formula (F2).
GYPD /G>1...(F1)
CYPD /C < 1...(F2)
G: β-glucan content (mass%) in the yeast cell wall
C: chitin content in the yeast cell wall (μmol/mg)
G YPD : β-glucan content (mass%) in yeast cell walls extracted from yeast cultured using YPD medium
C YPD : chitin content (μmol/mg) in yeast cell walls extracted from yeast cultured in YPD medium
　本発明の一態様によれば、免疫細胞の増殖、または、免疫を調節できる動物用飼料、食品素材、および酵母細胞壁の抽出方法を提供できる。 According to one aspect of the present invention, it is possible to provide animal feed, food materials, and a method for extracting yeast cell walls that can regulate the proliferation of immune cells or the immune system.
実施例１および比較例１で得られた酵母細胞壁（ＣＰおよびＹＰＤ）中のβ－グルカンの含量を示すグラフである。1 is a graph showing the β-glucan content in yeast cell walls (CP and YPD) obtained in Example 1 and Comparative Example 1. 実施例２および比較例２で得られた酵母細胞壁（ＳＰおよびＹＰＤ）中のβ－グルカンの含量を示すグラフである。1 is a graph showing the β-glucan content in yeast cell walls (SP and YPD) obtained in Example 2 and Comparative Example 2. 実施例１および比較例１で得られた酵母細胞壁（ＣＰおよびＹＰＤ）中のキチンの含量を示すグラフである。1 is a graph showing the chitin content in yeast cell walls (CP and YPD) obtained in Example 1 and Comparative Example 1. 実施例２および比較例２で得られた酵母細胞壁（ＳＰおよびＹＰＤ）中のキチンの含量を示すグラフである。1 is a graph showing the chitin content in yeast cell walls (SP and YPD) obtained in Example 2 and Comparative Example 2. 実施例１で得られた酵母細胞壁（Ｐ．Ｋｕｄｒｉａｖｚｅｖｉｉ）と、試薬（Ｃｏｎｃａｎａｖａｌｉｎ　ＡおよびＺｙｍｏｓａｎ）を用いて、末梢血単核細胞（ＰＢＭＣ）を生育したときの細胞増殖の結果を示すグラフである。This is a graph showing the results of cell proliferation when peripheral blood mononuclear cells (PBMCs) were grown using the yeast cell wall (P. Kudriavzevii) obtained in Example 1 and reagents (Concanavalin A and Zymosan). 実施例１で得られた酵母細胞壁（Ｐ．Ｋｕｄｒｉａｖｚｅｖｉｉ）と、酵母細胞壁の試薬（Ｚｙｍｏｓａｎ）を用いた場合におけるサイトカイン（腫瘍壊死因子、ＴＮＦａ）への影響を評価した結果を示すグラフである。1 is a graph showing the results of evaluating the effects on cytokines (tumor necrosis factor, TNFa) when the yeast cell wall (P. Kudriavzevii) obtained in Example 1 and a yeast cell wall reagent (Zymosan) are used. 実施例１で得られた酵母細胞壁（Ｐ．Ｋｕｄｒｉａｖｚｅｖｉｉ）と、酵母細胞壁の試薬（Ｚｙｍｏｓａｎ）を用いた場合におけるサイトカイン（インターロイキン－１β、ＩＬ－１ｂ）への影響を評価した結果を示すグラフである。1 is a graph showing the results of evaluating the effects on cytokines (interleukin-1β, IL-1b) when the yeast cell wall (P. Kudriavzevii) obtained in Example 1 and a yeast cell wall reagent (Zymosan) are used.
［動物用飼料］
　まず、本実施形態に係る動物用飼料について説明する。本実施形態に係る動物用飼料は、芋類からでんぷんを取り出した後の残渣である芋残渣を用いて培養した酵母から抽出した酵母細胞壁である動物用飼料である。
　そして、この酵母細胞壁中のβ－グルカンおよびキチンの含量を測定した場合に、下記数式（Ｆ１）および下記数式（Ｆ２）で示す両方の条件を満たすことが必要である。
ＧＹＰＤ／Ｇ　＞　１　・・・（Ｆ１）
ＣＹＰＤ／Ｃ　＜　１　・・・（Ｆ２）
Ｇ：前記酵母細胞壁中のβ－グルカンの含量（質量％）
Ｃ：前記酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
ＧＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のβ－グルカンの含量（質量％）
ＣＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ） [Animal feed]
First, the animal feed according to the present embodiment will be described. The animal feed according to the present embodiment is an animal feed made of yeast cell walls extracted from yeast cultured using potato residue, which is the residue remaining after extracting starch from potatoes.
When the contents of β-glucan and chitin in the yeast cell wall are measured, it is necessary that the conditions shown in the following formula (F1) and the following formula (F2) are both satisfied.
GYPD /G>1...(F1)
CYPD /C < 1...(F2)
G: β-glucan content (mass%) in the yeast cell wall
C: chitin content in the yeast cell wall (μmol/mg)
G YPD : β-glucan content (mass%) in yeast cell walls extracted from yeast cultured using YPD medium
C YPD : chitin content (μmol/mg) in yeast cell walls extracted from yeast cultured in YPD medium
　ＧＹＰＤ／Ｇの値が１以下の場合には、動物用飼料における免疫細胞の増殖効果が不十分となる。また、同様の観点から、ＧＹＰＤ／Ｇは、１．０５以上であることが好ましく、１．１以上であることがより好ましく、１．１９以上であることが特に好ましい。ＧＹＰＤ／Ｇの値の上限値は、特に制限されない。例えば、ＧＹＰＤ／Ｇの値は、１０以下であってもよく、５以下であってもよい。 When the value of G YPD /G is 1 or less, the immune cell proliferation effect in the animal feed becomes insufficient. From the same viewpoint, G YPD /G is preferably 1.05 or more, more preferably 1.1 or more, and particularly preferably 1.19 or more. The upper limit of the value of G YPD /G is not particularly limited. For example, the value of G YPD /G may be 10 or less, or 5 or less.
　なお、ＣＹＰＤ／Ｃの値は、０．９８以下であることが好ましく、０．９４以下であることがより好ましい。ＣＹＰＤ／Ｃの下限値は、特に制限されない。例えば、ＣＹＰＤ／Ｃの値は、０．１以上であってもよく、０．２以上であってもよい。 The value of C YPD /C is preferably 0.98 or less, more preferably 0.94 or less. The lower limit of C YPD /C is not particularly limited. For example, the value of C YPD /C may be 0.1 or more, or 0.2 or more.
　ＧＹＰＤ／Ｇの値とＣＹＰＤ／Ｃの値を前記範囲内に調整する方法としては、例えば、後述する本実施形態に係る酵母細胞壁の抽出方法を採用することが挙げられる。 An example of a method for adjusting the values of G YPD /G and C YPD /C to fall within the above ranges is to employ the yeast cell wall extraction method according to the present embodiment described below.
　本実施形態に係る動物用飼料は、乳牛、肉牛、羊、豚、鶏、および魚類からなる群から選択される少なくとも１つの動物に使用することが好ましい。 The animal feed of this embodiment is preferably used for at least one animal selected from the group consisting of dairy cows, beef cattle, sheep, pigs, chickens, and fish.
　本実施形態に用いる芋残渣は、芋類からでんぷんを取り出した後の残渣である。
　芋類としては、ジャガイモ、サツマイモ、キクイモ、アピオス、タロイモ、サトイモ、コンニャクイモ、ヤムイモ、キャッサバ、ナガイモ、およびヤマノイモなどが挙げられる。これらの中でも、原料入手の観点から、サツマイモ、またはキャッサバが好ましい。 The potato residue used in this embodiment is the residue remaining after starch is extracted from potatoes.
Examples of tubers include potato, sweet potato, Jerusalem artichoke, apios, taro, taro, konjac, yam, cassava, Chinese yam, and Chinese yam. Among these, sweet potato or cassava is preferred from the viewpoint of raw material availability.
　本実施形態に用いる酵母は、特に限定されない。酵母としては、ピキア・クドリアブゼビ（Ｐ．ｋｕｄｒｉａｖｚｅｖｉｉ）、サッカロミセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ）、およびクリベロミセス・マーキシアナス（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ　ｍａｒｘｉａｎｕｓ）などが挙げられる。 The yeast used in this embodiment is not particularly limited. Examples of yeast include Pichia kudriavzevii, Saccharomyces cerevisiae, and Kluyveromyces marxianus.
［酵母細胞壁の抽出方法］
　次に、本実施形態に係る酵母細胞壁の抽出方法について説明する。本実施形態に係る酵母細胞壁の抽出方法は、芋類からでんぷんを取り出した後の残渣である芋残渣を、でんぷん分解酵素と酵母とでエタノール発酵しつつ、前記酵母を培養して、エタノール発酵物を得る工程（以下、「第一工程」とも称する）と、前記エタノール発酵物から前記酵母を分離する工程（以下、「第二工程」とも称する）と、前記酵母から、下記条件１を満たす酵母細胞壁を抽出する工程（以下、「第三工程」とも称する）と、を備える方法である。
（条件１）
　前記酵母細胞壁中のβ－グルカンおよびキチンの含量を測定した場合に、前記数式（Ｆ１）および前記数式（Ｆ２）で示す両方の条件を満たす。 [Method for extracting yeast cell walls]
Next, the yeast cell wall extraction method according to the present embodiment will be described. The yeast cell wall extraction method according to the present embodiment includes a step of ethanol fermenting potato residue, which is the residue after extracting starch from potatoes, with starch-degrading enzymes and yeast while culturing the yeast to obtain an ethanol fermented product (hereinafter also referred to as the "first step"), a step of separating the yeast from the ethanol fermented product (hereinafter also referred to as the "second step"), and a step of extracting yeast cell walls from the yeast that satisfy the following condition 1 (hereinafter also referred to as the "third step").
(Condition 1)
When the contents of β-glucan and chitin in the yeast cell wall are measured, the conditions shown in both the formula (F1) and the formula (F2) are satisfied.
　本実施形態に係る酵母細胞壁の抽出方法によれば、前述の本実施形態に係る動物用飼料を効率よく、作製できる。 The yeast cell wall extraction method according to this embodiment allows the animal feed according to the embodiment described above to be produced efficiently.
　第一工程においては、まず、芋類からでんぷんを取り出した後の残渣である芋残渣を準備する。
　芋類としては、前述のとおりである。
　芋残渣の水分含有量は、特に限定されない。ただし、糖化発酵の観点からは、芋残渣の水分含有量は、３０質量％以上であることが好ましい。
　芋残渣としては、乾燥した芋残渣を使用してもよい。乾燥した芋残渣を使用する場合は、水を加え、芋残渣の水分含有量を調整すればよい。また、乾燥した芋残渣を、でんぷん搾取後の高水分含有の芋残渣に混合して使用してもよい。 In the first step, potato residue, which is the residue remaining after extracting starch from potatoes, is prepared.
As for potatoes, they are as mentioned above.
The water content of the potato residue is not particularly limited, however, from the viewpoint of saccharification and fermentation, the water content of the potato residue is preferably 30% by mass or more.
As the potato residue, dried potato residue may be used. When dried potato residue is used, water may be added to adjust the moisture content of the potato residue. Also, the dried potato residue may be mixed with the potato residue having a high moisture content after starch extraction.
　第一工程においては、次に、芋残渣を、でんぷん分解酵素と酵母とでエタノール発酵しつつ、酵母を培養して、エタノール発酵物を得る。
　芋残渣は、そのまま発酵処理してもよいが、芋残渣に残存するでんぷんを糊化するため、発酵処理の前に加熱処理することが好ましい。加熱条件としては、加熱温度が６０℃以上であることが好ましく、加熱時間が５分間以上であることが好ましい。加熱温度が６０℃以上であれば、芋残渣に残存するでんぷんの糊化効率を高めることができる。加熱時間は、特に限定されず、加熱温度を考慮して適宜設定できる。 In the first step, the potato residue is then subjected to ethanol fermentation using starch-degrading enzymes and yeast, while the yeast is cultured, to obtain an ethanol fermented product.
The potato residue may be fermented as is, but it is preferable to heat treat it before fermentation in order to gelatinize the starch remaining in the potato residue. As heating conditions, a heating temperature of 60°C or higher and a heating time of 5 minutes or longer are preferable. A heating temperature of 60°C or higher can increase the gelatinization efficiency of the starch remaining in the potato residue. The heating time is not particularly limited and can be set appropriately taking into account the heating temperature.
　でんぷん分解酵素による処理と、酵母による発酵処理とは、別々に行ってもよく、同時に行ってもよい。 The treatment with the starch-degrading enzyme and the fermentation treatment with yeast can be carried out separately or simultaneously.
　酵母は、前述のとおりである。
　でんぷん分解酵素は、特に限定されるものではなく、でんぷんを分解する酵素であればよい。でんぷん分解酵素としては、α－アミラーゼ、およびグルコアミラーゼなどが挙げられる。また、芋残渣の粘性を下げるために、でんぷん分解酵素以外に、ペクチナーゼ、またはセルラーゼなどを併用してもよい。 The yeast is as described above.
The starch-degrading enzyme is not particularly limited, and may be any enzyme that degrades starch. Examples of the starch-degrading enzyme include α-amylase and glucoamylase. In order to reduce the viscosity of the potato residue, pectinase or cellulase may be used in combination with the starch-degrading enzyme.
　α－アミラーゼの添加量は、芋残渣固形分１ｇ当たり、９×１０－５ユニット（以下、「Ｕ」とも称する）以上６００Ｕ以下であることが好ましく、８×１０－３Ｕ以上２０Ｕ以下であることがより好ましい。グルコアミラーゼは、芋残渣固形分１ｇ当たり、３×１０－４Ｕ以上２００Ｕ以下であることが好ましく、３×１０－２Ｕ以上０．２Ｕ以下であることがより好ましい。
　セルラーゼを用いる場合、その添加量は、芋残渣固形分１ｇ当たり、１×１０－４Ｕ以上１００Ｕ以下で添加することが好ましい。ペクチナーゼを用いる場合、その添加量は、芋残渣固形分１ｇ当たり、１×１０－３Ｕ以上１０００Ｕ以下であることが好ましく、１×１０－１Ｕ以上１Ｕ以下であることがより好ましい。 The amount of α-amylase added is preferably 9×10 −5 units (hereinafter also referred to as “U”) to 600 U per gram of potato residue solid content, and more preferably 8×10 −3 U to 20 U. The amount of glucoamylase added is preferably 3×10 −4 U to 200 U per gram of potato residue solid content, and more preferably 3×10 −2 U to 0.2 U.
When cellulase is used, the amount added is preferably 1×10 −4 U to 100 U per gram of potato residue solid content. When pectinase is used, the amount added is preferably 1×10 −3 U to 1000 U, more preferably 1×10 −1 U to 1 U, per gram of potato residue solid content.
　第二工程においては、エタノール発酵物から酵母を分離する。
　エタノール発酵物に含まれるエタノールなどの液体成分は、エタノール発酵物を圧搾する方法、および、エタノール発酵物を加熱する方法の少なくとも一方を行うことにより、除去できる。
　エタノール発酵物から液体成分を除去すれば、酵母を分離できる。 In the second step, the yeast is separated from the ethanol fermentate.
Liquid components such as ethanol contained in the ethanol fermentation product can be removed by at least one of a method of squeezing the ethanol fermentation product and a method of heating the ethanol fermentation product.
By removing the liquid components from the ethanol fermentation product, the yeast can be separated.
　第三工程においては、酵母から、酵母細胞壁を抽出する。
　酵母細胞壁を抽出するためには、まず、温度５０℃で１０時間以上インキュベートすることで自己融解を生じさせた後、酵母を粉砕して、粉砕物を得ることが好ましい。
　粉砕方法は、乾式粉砕であってよく、湿式粉砕であってもよい。粉砕装置としては、ビーズミル、ボールミル、およびコロイドミルなどが挙げられる。
　そして、得られた粉砕物を、エタノールなどで洗浄し、その後、固形分を沈殿させることで、酵母細胞壁を画分して、酵母細胞壁を抽出できる。
　以上のようにして、前記条件１を満たす酵母細胞壁を抽出できる。 In the third step, the yeast cell walls are extracted from the yeast.
In order to extract the yeast cell walls, it is preferable to first cause autolysis by incubating at a temperature of 50° C. for 10 hours or more, and then pulverize the yeast to obtain a pulverized product.
The pulverization method may be dry pulverization or wet pulverization. Examples of the pulverization apparatus include a bead mill, a ball mill, and a colloid mill.
The resulting pulverized product is washed with ethanol or the like, and then the solid content is precipitated to fractionate the yeast cell walls, allowing the yeast cell walls to be extracted.
In this manner, yeast cell walls that satisfy condition 1 above can be extracted.
［食品素材］
　次に、本実施形態に係る食品素材について説明する。本実施形態に係る食品素材は、前述の本実施形態に係る動物用飼料と同様のものである。
　前述のとおり、本実施形態に係る動物用飼料は、免疫細胞の増殖、または、免疫を調節できる。そのため、食品素材としても、健康増進などの効果が見込める。 [Food ingredients]
Next, the food material according to the present embodiment will be described. The food material according to the present embodiment is the same as the animal feed according to the present embodiment described above.
As described above, the animal feed according to this embodiment can regulate the proliferation of immune cells or the immune system. Therefore, as a food ingredient, it is expected to have effects such as health promotion.
　次に、本発明を実施例および比較例によりさらに詳細に説明するが、本発明はこれらの例によってなんら限定されるものではない。 Next, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples in any way.
［実施例１］
　乾燥したキャッサバ残渣２０ｇと純水８０ｇを混合して、水分含有量８０質量％のキャッサバ残渣を得た。得られたキャッサバ残渣に、キャッサバ残渣固形分１ｇあたり、８～１０ユニットのα－アミラーゼを添加し、温度３０～１００℃、０．４～１時間の条件で加熱処理を行い、その後、冷却して、培地を得た。
　得られた培地に、酵素カクテル（グルコアミラーゼ１．２８ユニット、ペクチナーゼ４．０ユニット、セルラーゼ０．４６ユニット）、および窒素源（酵母エキス＋ペプトン）を添加し、さらに、酵母としてピキア・クドリアブゼビ（Ｐ．ｋｕｄｒｉａｖｚｅｖｉｉ）を接種して、温度３０～４０℃、攪拌回転数５０～１５０ｒｐｍ、２４～４０時間の条件で酵母を培養して、エタノール発酵物を得た。
　次に、得られたエタノール発酵物を圧搾して、エタノールを含む液体を除去して、酵母を分離した。そして、分離した酵母を、自己融解してビーズミルを用いて、粉砕した後に、エタノールで洗浄し、その後、固形分を沈殿させることで、酵母細胞壁を画分して、酵母細胞壁を抽出した。 [Example 1]
20 g of dried cassava residue was mixed with 80 g of pure water to obtain a cassava residue with a moisture content of 80% by mass. 8 to 10 units of α-amylase per 1 g of cassava residue solid content was added to the obtained cassava residue, and the mixture was heat-treated at a temperature of 30 to 100° C. for 0.4 to 1 hour, and then cooled to obtain a medium.
To the obtained medium, an enzyme cocktail (glucoamylase 1.28 units, pectinase 4.0 units, cellulase 0.46 units) and a nitrogen source (yeast extract + peptone) were added, and further, Pichia kudriavzevii was inoculated as a yeast. The yeast was cultured under conditions of a temperature of 30 to 40°C, an agitation speed of 50 to 150 rpm, and for 24 to 40 hours to obtain an ethanol fermentation product.
Next, the obtained ethanol fermentation product was squeezed to remove the liquid containing ethanol, and the yeast was separated. The separated yeast was then autolyzed and crushed using a bead mill, washed with ethanol, and the solids were then precipitated to fractionate and extract the yeast cell walls.
［実施例２］
　乾燥したキャッサバ残渣２０ｇに代えて、乾燥したサツマイモ残渣２０ｇを用いた以外は、実施例１と同様にして、培地および酵母細胞壁を得た。 [Example 2]
The medium and yeast cell walls were obtained in the same manner as in Example 1, except that 20 g of dried sweet potato residue was used instead of 20 g of dried cassava residue.
［比較例１］
　培地として、ＹＰＤ培地を用いた以外は、実施例１と同様にして、酵母細胞壁を得た。 [Comparative Example 1]
Yeast cell walls were obtained in the same manner as in Example 1, except that YPD medium was used as the medium.
［比較例２］
　培地として、ＹＰＤ培地を用いた以外は、実施例２と同様にして、酵母細胞壁を得た。 [Comparative Example 2]
Yeast cell walls were obtained in the same manner as in Example 2, except that YPD medium was used as the medium.
［酵母細胞壁中のβ－グルカンおよびキチンの含量の測定］
　実施例１、実施例２、比較例１および比較例２で得られた酵母細胞壁中のβ－グルカンおよびキチンの含量を、次のようにして、測定した。
　β－グルカンは、Ｍｅｇａｚｙｍｅ社製のＥｎｚｙｍａｔｉｃ　ｙｅａｓｔ　ｂｅｔａ　ｇｌｕｃａｎ　ａｓｓａｙ ｐｒｏｃｅｄｕｒｅ（Ｋ－ＥＢＨＬＧ）を用いて測定した。
　キチンは、下記参考文献１および２に従って、ＭＢＴＨ（３－ｍｅｔｈｙｌ－ｂｅｎｚｏｔｈｉａｚｏｌｉｎｏｎｅ　ｈｙｄｒａｚｏｎｅ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ）法を用いて測定した。
　参考文献１：Ｔｓｕｊｉ　Ａ，　Ｋｉｎｏｓｈｉｔａ　Ｔ，　Ｈｏｓｈｉｎｏ　Ｍ．　１９６９．　Ａｎａｌｙｔｉｃａｌ　ｃｈｅｍｉｃａｌ　ｓｔｕｄｉｅｓ　ｏｎ　ａｍｉｎｏ　ｓｕｇａｒｓ．　ＩＩ．　Ｄｅｔｅｒｍｉｎａｔｉｏｎ　ｏｆ　ｈｅｘｏｓａｍｉｎｅｓ　ｕｓｉｎｇ　３－ｍｅｔｈｙｌ－２－ｂｅｎｚｏｔｈｉａｚｏｌｏｎｅ　ｈｙｄｒａｚｏｎｅ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ．　Ｃｈｅｍ　Ｐｈａｒｍ　Ｂｕｌｌ　（Ｔｏｋｙｏ）１７：１５０５－１５１０．
　参考文献２：Ｔｓｕｊｉ　Ａ，　Ｋｉｎｏｓｈｉｔａ　Ｔ，　Ｈｏｓｈｉｎｏ　Ｍ．　１９６９．　Ｍｉｃｒｏｄｅｔｅｒｍｉｎａｔｉｏｎ　ｏｆ　ｈｅｘｏｓａｍｉｎｅｓ．　Ｃｈｅｍ　Ｐｈａｒｍ　Ｂｕｌｌ　（Ｔｏｋｙｏ）　１７：２１７－２１８． [Measurement of β-glucan and chitin contents in yeast cell walls]
The contents of β-glucan and chitin in the yeast cell walls obtained in Examples 1 and 2 and Comparative Examples 1 and 2 were measured as follows.
β-Glucan was measured using the Enzymatic Yeast Beta Glucan Assay Procedure (K-EBHLG) manufactured by Megazyme.
Chitin was measured using the MBTH (3-methyl-benzothiazolinone hydrozone hydrochloride) method according to References 1 and 2 below.
Reference 1: Tsuji A, Kinoshita T, Hoshino M. 1969. Analytical chemical studies on amino sugars. II. Determination of hexosamines using 3-methyl-2-benzothiazolone hydrazone hydrochloride. Chem Pharm Bull (Tokyo) 17:1505-1510.
Reference 2: Tsuji A, Kinoshita T, Hoshino M. 1969. Microdetermination of hexosamines. Chem Pharm Bull (Tokyo) 17:217-218.
　実施例１および比較例１で得られた酵母細胞壁中のβ－グルカンの含量を、図１に示す。また、実施例２および比較例２で得られた酵母細胞壁中のβ－グルカンの含量を、図２に示す。
　実施例１および比較例１で得られた酵母細胞壁中のキチンの含量を、図３に示す。実施例２および比較例２で得られた酵母細胞壁中のキチンの含量を、図４に示す。
　なお、図１～図４において、実施例１は、キャッサバ残渣を原料とする培地を用いているので、ＣＰと表記し、実施例２は、サツマイモ残渣を原料とする培地を用いているので、ＳＰと表記した。また、比較例１および比較例２は、ＹＰＤ培地を用いているので、ＹＰＤと表記した。
　また、図１、図２および図４では、エラーバーで結果を表記した。図３では、１回目の試験結果を左側に記載し、２回目の試験結果を右側に記載した。 The β-glucan contents in the yeast cell walls obtained in Example 1 and Comparative Example 1 are shown in Figure 1. The β-glucan contents in the yeast cell walls obtained in Example 2 and Comparative Example 2 are shown in Figure 2.
The chitin contents in the yeast cell walls obtained in Example 1 and Comparative Example 1 are shown in Figure 3. The chitin contents in the yeast cell walls obtained in Example 2 and Comparative Example 2 are shown in Figure 4.
1 to 4, Example 1 uses a medium made of cassava residue as a raw material, and is therefore indicated as CP, and Example 2 uses a medium made of sweet potato residue as a raw material, and is therefore indicated as SP. Comparative Examples 1 and 2 use a YPD medium, and are therefore indicated as YPD.
Moreover, the results are indicated by error bars in Figures 1, 2, and 4. In Figure 3, the results of the first test are shown on the left side, and the results of the second test are shown on the right side.
　図１および図３に示す結果から、実施例１で得られた酵母細胞壁においては、ＧＹＰＤ／Ｇの値が１超であり、ＣＹＰＤ／Ｃの値が１未満であることが確認された。
　また、図２および図４に示す結果から、実施例２で得られた酵母細胞壁においては、ＧＹＰＤ／Ｇの値が１超であり、ＣＹＰＤ／Ｃの値が１未満であることが確認された。 1 and 3, it was confirmed that the yeast cell wall obtained in Example 1 had a G YPD /G value of more than 1 and a C YPD /C value of less than 1.
2 and 4, it was confirmed that the yeast cell wall obtained in Example 2 had a G YPD /G value of more than 1 and a C YPD /C value of less than 1.
［末梢血単核細胞の生育の評価］
　実施例１で得られた酵母細胞壁、Ｃｏｎｃａｎａｖａｌｉｎ　Ａ（富士フイルム和光純薬株式会社製）、およびＺｙｍｏｓａｎ（富士フイルム和光純薬株式会社製）について、これらの末梢血単核細胞（以下、「ＰＢＭＣ」とも称する）の生育への影響を、以下のようにして評価した。
　すなわち、ウシ血液からＰＢＭＣを分離し、培養したものを試料とした。この試料に、酵母細胞壁や試薬を添加し、温度３７℃、７２時間の条件で、ＰＢＭＣを増殖させた。そして、増殖後の試料について、Ｃｅｌｌ　Ｃｏｕｎｔｉｎｇ　Ｋｉｔ　８（同仁化学研究所社製）を加えて４５０ｎｍの吸光度（Ａ４５０）を測定することで、ＰＢＭＣの生育を評価した。 [Evaluation of peripheral blood mononuclear cell growth]
The yeast cell wall obtained in Example 1, Concanavalin A (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), and Zymosan (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were evaluated for their effects on the growth of peripheral blood mononuclear cells (hereinafter also referred to as "PBMC") as follows.
That is, PBMCs were isolated from bovine blood and cultured to prepare samples. Yeast cell walls and reagents were added to the samples, and the PBMCs were grown at 37° C. for 72 hours. After growth, Cell Counting Kit 8 (Dojindo Laboratories) was added to the samples, and the absorbance at 450 nm (A450) was measured to evaluate the growth of PBMCs.
　得られた結果を、図５に示す。なお、図５において、実施例１は、酵母としてピキア・クドリアブゼビを用いているので、Ｐ．ｋｕｄｒｉａｖｚｅｖｉｉと表記し、試料に酵母細胞壁や試薬を添加しなかった例はＣｏｎｔｒｏｌと表記し、それ以外は、それぞれ試薬名の略称を表記した。なお、略称は、以下のとおりである。
　ＣｏｎＡ：　Ｃｏｎｃａｎａｖａｌｉｎ　Ａ（３μｇ／ｍＬ）
　Ｚｙｍｏｓａｎ：　β－ｇｌｕｃａｎ　標品（１００ｕｇ／ｍＬ）
　ＣＰ：キャッサバ残渣
　図５に示す結果から、実施例１で得られた酵母細胞壁は、酵母細胞壁の試薬であるＺｙｍｏｓａｎと比較して、ＰＢＭＣの増殖効果がかなり高いことが確認された。また、実施例１で得られた酵母細胞壁は、免疫細胞の活性化試薬であるＣｏｎｃａｎａｖａｌｉｎ　Ａと比較して、ＰＢＭＣの増殖効果が高いことが確認された。 The results are shown in Figure 5. In Figure 5, Example 1 uses Pichia kudriavzevii as yeast, and is therefore indicated as P. kudriavzevii, and the example in which no yeast cell wall or reagent was added to the sample is indicated as Control, and the rest are indicated by the abbreviations of the reagent names. The abbreviations are as follows:
ConA: Concanavalin A (3μg/mL)
Zymosan: β-glucan standard (100ug/mL)
CP: Cassava residue From the results shown in Figure 5, it was confirmed that the yeast cell wall obtained in Example 1 has a significantly higher PBMC proliferation effect than Zymosan, a yeast cell wall reagent. It was also confirmed that the yeast cell wall obtained in Example 1 has a higher PBMC proliferation effect than Concanavalin A, an immune cell activation reagent.
［サイトカインへの影響の評価］
　実施例１で得られた酵母細胞壁、およびＺｙｍｏｓａｎ（富士フイルム和光純薬株式会社製）について、これらのサイトカインへの影響を、以下のようにして評価した。なお、サイトカインとして、腫瘍壊死因子（ＴＮＦα）と、インターロイキン－１β（ＩＬ－１ｂ）について、評価を行った。
　すなわち、ウシ血液から分離したＰＢＭＣに酵母細胞壁およびＺｙｍｏｓａｎを添加し、温度３７℃、６時間の条件で刺激を行い、ＲＮＡを抽出した後に定量ＰＣＲ法にて標準遺伝子であるβ－Ａｃｔｉｎに対する各サイトカインの遺伝子発現量を測定することで、酵母細胞壁のサイトカイン産生に対する影響を評価した。 [Evaluation of effects on cytokines]
The yeast cell wall obtained in Example 1 and Zymosan (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were evaluated for their effects on cytokines as follows. As cytokines, tumor necrosis factor (TNFα) and interleukin-1β (IL-1b) were evaluated.
That is, yeast cell walls and Zymosan were added to PBMCs isolated from bovine blood, and the PBMCs were stimulated at 37°C for 6 hours. RNA was then extracted, and the gene expression level of each cytokine relative to the standard gene β-Actin was measured by quantitative PCR to evaluate the effect of the yeast cell walls on cytokine production.
　実施例１で得られた酵母細胞壁と、酵母細胞壁の試薬を用いた場合における腫瘍壊死因子（ＴＮＦα）への影響を評価した結果を、図６に示す。
　実施例１で得られた酵母細胞壁と、酵母細胞壁の試薬を用いた場合におけるインターロイキン－１β（ＩＬ－１ｂ）への影響を評価した結果を、図７に示す。
　なお、図６および図７において、実施例１は、酵母としてピキア・クドリアブゼビを用いているので、Ｐ．ｋｕｄｒｉａｖｚｅｖｉｉと表記し、Ｚｙｍｏｓａｎは、試薬名を表記した。また、酵母細胞壁を使用しなかった例は、Ｃｏｎｔｒｏｌと表記した。 FIG. 6 shows the results of evaluating the effects of the yeast cell wall obtained in Example 1 and the yeast cell wall reagent on tumor necrosis factor (TNFα).
FIG. 7 shows the results of evaluating the effect on interleukin-1β (IL-1b) when the yeast cell wall obtained in Example 1 and a yeast cell wall reagent are used.
6 and 7, Example 1 uses Pichia kudriavzevii as yeast, and is therefore indicated as P. kudriavzevii, and Zymosan is the name of the reagent. In addition, the example in which the yeast cell wall was not used is indicated as Control.
　図６および図７に示す結果から、実施例１で得られた酵母細胞壁は、酵母細胞壁の試薬であるＺｙｍｏｓａｎと比較して、遺伝子発現が抑制できることが確認された。また、実施例１で得られた酵母細胞壁は、酵母細胞壁を使用しなかった場合（Ｃｏｎｔｒｏｌ）と比較して、遺伝子発現が抑制できることが確認された。よって、実施例１で得られた酵母細胞壁によれば、免疫を調節できることが確認された。 The results shown in Figures 6 and 7 confirm that the yeast cell wall obtained in Example 1 is able to suppress gene expression, compared to Zymosan, a yeast cell wall reagent. It was also confirmed that the yeast cell wall obtained in Example 1 is able to suppress gene expression, compared to a case in which no yeast cell wall was used (Control). Thus, it was confirmed that the yeast cell wall obtained in Example 1 is able to regulate immunity.

Claims (6)

  1. 　芋類からでんぷんを取り出した後の残渣である芋残渣を用いて培養した酵母から抽出した酵母細胞壁である動物用飼料であって、
    　前記酵母細胞壁中のβ－グルカンおよびキチンの含量を測定した場合に、下記数式（Ｆ１）および下記数式（Ｆ２）で示す両方の条件を満たす、
    　動物用飼料。
    ＧＹＰＤ／Ｇ　＞　１　・・・（Ｆ１）
    ＣＹＰＤ／Ｃ　＜　１　・・・（Ｆ２）
    Ｇ：前記酵母細胞壁中のβ－グルカンの含量（質量％）
    Ｃ：前記酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
    ＧＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のβ－グルカンの含量（質量％）
    ＣＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）     An animal feed comprising yeast cell walls extracted from yeast cultured using potato residue, which is the residue left after extracting starch from potatoes,
    When the contents of β-glucan and chitin in the yeast cell wall are measured, both of the conditions shown in the following formula (F1) and the following formula (F2) are satisfied.
    Animal feed.
    GYPD /G>1...(F1)
    CYPD /C < 1...(F2)
    G: β-glucan content (mass%) in the yeast cell wall
    C: chitin content in the yeast cell wall (μmol/mg)
    G YPD : β-glucan content (mass%) in yeast cell walls extracted from yeast cultured using YPD medium
    C YPD : chitin content (μmol/mg) in yeast cell walls extracted from yeast cultured in YPD medium
  2. 　請求項１に記載の動物用飼料において、
    　前記ＧＹＰＤ／Ｇの値が、１．１９以上であり、
    　前記ＣＹＰＤ／Ｃの値が、０．９８以下である、
    　動物用飼料。     The animal feed according to claim 1,
    the value of G YPD /G is 1.19 or more;
    The C YPD /C value is 0.98 or less.
    Animal feed.
  3. 　請求項１または請求項２に記載の動物用飼料において、
    　前記芋類が、ジャガイモ、サツマイモ、キクイモ、アピオス、タロイモ、サトイモ、コンニャクイモ、ヤムイモ、キャッサバ、ナガイモ、およびヤマノイモからなる群から選択される少なくとも１つである、
    　動物用飼料。     In the animal feed according to claim 1 or 2,
    The tuber is at least one selected from the group consisting of potato, sweet potato, Jerusalem artichoke, apios, taro, taro, konjac, yam, cassava, Chinese yam, and Chinese yam;
    Animal feed.
  4. 　請求項１または請求項２に記載の動物用飼料において、
    　前記動物が、乳牛、肉牛、羊、豚、鶏、および魚類からなる群から選択される少なくとも１つである、
    　動物用飼料。     In the animal feed according to claim 1 or 2,
    The animal is at least one selected from the group consisting of dairy cattle, beef cattle, sheep, pigs, chickens, and fish;
    Animal feed.
  5. 　芋類からでんぷんを取り出した後の残渣である芋残渣を用いて培養した酵母から抽出した酵母細胞壁である食品素材であって、
    　前記酵母細胞壁中のβ－グルカンおよびキチンの含量を測定した場合に、下記数式（Ｆ１）および下記数式（Ｆ２）で示す両方の条件を満たす、
    　食品素材。
    ＧＹＰＤ／Ｇ　＞　１　・・・（Ｆ１）
    ＣＹＰＤ／Ｃ　＜　１　・・・（Ｆ２）
    Ｇ：前記酵母細胞壁中のβ－グルカンの含量（質量％）
    Ｃ：前記酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
    ＧＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のβ－グルカンの含量（質量％）
    ＣＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）     A food material which is a yeast cell wall extracted from yeast cultured using potato residue, which is the residue after extracting starch from potatoes,
    When the contents of β-glucan and chitin in the yeast cell wall are measured, both of the conditions shown in the following formula (F1) and the following formula (F2) are satisfied.
    Food ingredients.
    GYPD /G>1...(F1)
    CYPD /C < 1...(F2)
    G: β-glucan content (mass%) in the yeast cell wall
    C: chitin content in the yeast cell wall (μmol/mg)
    G YPD : β-glucan content (mass%) in yeast cell walls extracted from yeast cultured using YPD medium
    C YPD : chitin content (μmol/mg) in yeast cell walls extracted from yeast cultured in YPD medium
  6. 　芋類からでんぷんを取り出した後の残渣である芋残渣を、でんぷん分解酵素と酵母とでエタノール発酵しつつ、前記酵母を培養して、エタノール発酵物を得る工程と、
    　前記エタノール発酵物から前記酵母を分離する工程と、
    　前記酵母から、下記条件１を満たす酵母細胞壁を抽出する工程と、を備える、
    　酵母細胞壁の抽出方法。
    （条件１）
    　前記酵母細胞壁中のβ－グルカンおよびキチンの含量を測定した場合に、下記数式（Ｆ１）および下記数式（Ｆ２）で示す両方の条件を満たす。
    ＧＹＰＤ／Ｇ　＞　１　・・・（Ｆ１）
    ＣＹＰＤ／Ｃ　＜　１　・・・（Ｆ２）
    Ｇ：前記酵母細胞壁中のβ－グルカンの含量（質量％）
    Ｃ：前記酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）
    ＧＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のβ－グルカンの含量（質量％）
    ＣＹＰＤ：ＹＰＤ培地を用いて培養した酵母から抽出した酵母細胞壁中のキチンの含量（μｍｏｌ／ｍｇ）     A step of fermenting potato residue, which is the residue after removing starch from potatoes, into ethanol using a starch-degrading enzyme and yeast while culturing the yeast to obtain an ethanol fermented product;
    Separating the yeast from the ethanol fermentation product;
    The method includes a step of extracting a yeast cell wall that satisfies the following condition 1 from the yeast.
    Methods for extracting yeast cell walls.
    (Condition 1)
    When the contents of β-glucan and chitin in the yeast cell wall are measured, the contents satisfy both the conditions shown in the following formula (F1) and the following formula (F2).
    GYPD /G>1...(F1)
    CYPD /C < 1...(F2)
    G: β-glucan content (mass%) in the yeast cell wall
    C: chitin content in the yeast cell wall (μmol/mg)
    G YPD : β-glucan content (mass%) in yeast cell walls extracted from yeast cultured using YPD medium
    C YPD : chitin content (μmol/mg) in yeast cell walls extracted from yeast cultured in YPD medium
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