JP2998124B2 - Hydrophobicity measurement method and measurement kit - Google Patents
Hydrophobicity measurement method and measurement kitInfo
- Publication number
- JP2998124B2 JP2998124B2 JP12214396A JP12214396A JP2998124B2 JP 2998124 B2 JP2998124 B2 JP 2998124B2 JP 12214396 A JP12214396 A JP 12214396A JP 12214396 A JP12214396 A JP 12214396A JP 2998124 B2 JP2998124 B2 JP 2998124B2
- Authority
- JP
- Japan
- Prior art keywords
- yeast
- hydrophobicity
- measurement
- measuring
- column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Description
【０００１】[0001]
【発明の属する技術分野】本発明は細胞表層の疎水度測
定方法およびその測定キットに関する。The present invention relates to a method for measuring the hydrophobicity of a cell surface layer and a kit for measuring the same.
【０００２】[0002]
【従来の技術】酵母の一般的な凝集能測定として知られ
ている方法として、Ｂｕｒｎｓ法（Ｊ．Ｉｎｓｔ．Ｂｒ
ｅｗ．，４３，３１，１９３７）、Ｈｅｌｍ法（Ｗａｌ
ｌｅｒｓｔｅｉｎ Ｌａｂｏｒａｔｏｒｙ Ｃｏｍｍｕ
ｎｉｃａｔｉｏｎｓ１６，３１５，１９５３）およびそ
の改良法がある。近年、下面ビール酵母の凝集性株と非
凝集性株では、細胞表層の疎水性に差異があり、疎水性
と凝集性に関連があることが報告されている。 （Ａｐｐｌ．Ｅｎｖｉｒｏｎ．Ｍｉｃｒｏｂｉｏｌ．，
５８，３７０９，１９９２ Ｙｅａｓｔ １２，２０７
−２１３，１９９６） （Ｙｅａｓｔ １２，２０７−２１３，１９９６）で
は、酵母懸濁液中の疎水性細胞をポリスチレンでコーテ
ィングした常磁性のラテックスビーズに付着させ、これ
をマグネットにより分離することで酵母の疎水度を測定
するｍａｇｎｏｂｅａｄ法が開示されている。一般に酵
母や細菌などの疎水度を測定する方法として、ｃｏｎｔ
ａｃｔ ａｎｇｌｅ法（Ｊ．Ｍｉｃｒｏｂｉｏｌ．Ｍｅ
ｔｈｏｄｓ ６，９９−１１２，１９８７）、有機溶媒
との混合反応を見る方法（ＦＥＭＳ Ｍｉｃｒｏｂｉｏ
ｌ．Ｌｅｔｔ．９，２９−３３）が知られており、カラ
ムを用いる方法としてはＨＩＣ法（ＢＩＯＣＨＩＭＩＣ
Ａ ＥＴ ＢＩＯＰＨＹＳＩＣＡ ＡＣＴＡ．９４５３
２４−３３４ １９８８）等が開示されている。2. Description of the Related Art As a method known as a general measurement of aggregating ability of yeast, the Burns method (J. Inst. Br) is known.
ew. , 43, 31, 1937), the Helm method (Wal
lerstein Laboratory Commu
nations 16, 315, 1953) and improvements thereof. In recent years, it has been reported that there is a difference in the hydrophobicity of the cell surface between the flocculent strain and the non-coagulable strain of bottom beer yeast, and that the hydrophobicity and the flocculation property are related. (Appl. Environ. Microbiol.,
58, 3709, 1992 Yeast 12, 207
In (Yeast 12, 207-213, 1996), hydrophobic cells in a yeast suspension were attached to paramagnetic latex beads coated with polystyrene and separated by a magnet to separate yeast cells. A magnobead method for measuring hydrophobicity is disclosed. In general, as a method for measuring the hydrophobicity of yeast or bacteria, cont
act angle method (J. Microbiol. Me
methods 6,99-112,1987), a method of observing a mixed reaction with an organic solvent (FEMS Microbio)
l. Lett. 9, 29-33) are known, and as a method using a column, the HIC method (BIOCHIMIC) is used.
A ET BIOPHYSICA ACTA. 9453
24-334, 1988).
【０００３】[0003]
【発明が解決しようとする課題】ビール醸造において、
下面酵母の凝集能を測定することは使用酵母の選択に始
まり、発酵管理や最終製品の品質管理を行う上で重要で
ある。現在、ビール酵母の凝集性を測定する方法とし
て、Ｂｕｒｎｓ法、Ｈｅｌｍ法が一般的な凝集能測定法
として知られているが、凝集能の小さな変化や凝集性の
異なる酵母が混在している試料の凝集能をとらえにくい
という課題がある。最近開示されたｍａｇｎｏｂｅａｄ
法では、測定に使用するマグネットビーズが一般的では
なく、手に入りにくいという欠点がある。また、本測定
法によって得られた疎水度と凝集能の相関についてはあ
まり検討が進められていない。SUMMARY OF THE INVENTION In beer brewing,
Measuring the agglutinating ability of the bottom yeast starts with the selection of the yeast to be used, and is important for fermentation management and quality control of the final product. At present, as methods for measuring the cohesiveness of brewer's yeast, the Burns method and the Helm method are known as general methods for measuring cohesiveness. However, there is a problem that it is difficult to capture the cohesive ability of the particles. Recently disclosed magnetobeads
The method has the disadvantage that magnet beads used for measurement are not common and are difficult to obtain. Further, the correlation between the hydrophobicity obtained by this measurement method and the aggregation ability has not been studied much.
【０００４】更に、一般に細胞の疎水性を測定するｃｏ
ｎｔａｃｔ ａｎｇｌｅ法では、顕微鏡に装着するゴニ
オメトリーという特別な装置が必要であり、操作は煩雑
な上、再現性が低い。また有機溶媒との混合反応を見る
方法は、精度・再現性の点で問題が多い。またカラムを
用いて疎水度を測定するＨＩＣ法は、細菌や酵母等につ
いて測定できるとされているが感度は低く、測定にあた
り４種類のカラムおよび溶出液を調整して使用しなけれ
ばならないことや細胞にとってダメージの大きい測定系
でもあり問題がある。[0004] Furthermore, in general, co-
In the nact angle method, a special device called goniometry to be attached to a microscope is required, and the operation is complicated and the reproducibility is low. Also, the method of observing a mixed reaction with an organic solvent has many problems in terms of accuracy and reproducibility. The HIC method, which measures the hydrophobicity using a column, is said to be able to measure bacteria, yeasts, etc., but the sensitivity is low, and it is necessary to adjust and use four types of columns and eluate for measurement. It is also a measurement system with large damage to cells, and has a problem.
【０００５】そこで本発明は、近年報告されている酵母
の凝集性と細胞表面疎水度の関係に着目して、簡便な操
作で精度よく酵母表層の疎水性を測定でき、なおかつそ
の凝集能が推定できる方法および測定キットを提供する
ことを目的とするものである。Therefore, the present invention focuses on the recently reported relationship between the agglutinability of yeast and the hydrophobicity of the cell surface, whereby the hydrophobicity of the yeast surface layer can be accurately measured by a simple operation, and the agglutination ability is estimated. An object of the present invention is to provide a method and a measurement kit which can be used.
【０００６】[0006]
【課題を解決するための手段】本発明者は、上記課題を
検討した結果、簡易なカラム装置と特定の緩衝液を利用
することにより、従来の方法よりも簡便な操作で精度良
く酵母表層の疎水性の測定を行い、酵母の凝集能の変化
や凝集性の異なる酵母の混合試料の凝集能が推定できる
疎水度測定法及びその測定キットを見出し、本発明を完
成した。更に本発明は、酵母だけでなく、細菌等の細胞
の疎水度測定にも利用できることを見出した。Means for Solving the Problems As a result of studying the above-mentioned problems, the present inventor has found that the use of a simple column apparatus and a specific buffer allows the yeast surface layer to be more accurately formed by a simpler operation than the conventional method. The present inventors have completed the present invention by measuring the hydrophobicity and finding a method for measuring the degree of hydrophobicity and a kit for measuring the degree of hydrophobicity, which are capable of estimating the change in the aggregating ability of yeast and the aggregating ability of mixed samples of yeasts having different agglutinability. Furthermore, it has been found that the present invention can be used not only for measuring the hydrophobicity of cells such as bacteria but also yeast.
【０００７】すなわち、本発明の第１は、塩を含む緩衝
液で平衡化したゲルを充填したカラムに対する細胞の吸
着率により、細胞表層の疎水度を測定する方法に関す
る。本発明の第２は、細胞が酵母、細菌である前記の測
定方法に関する。本発明の第３は、塩を含むｐＨ４．２
に調整したＡｃａｔａｔｅ Ｂｕｆｆｅｒで平衡化した
疎水性ゲルを充填したカラムに対するビール酵母の吸着
率により、ビール酵母の疎水度を測定する方法に関す
る。本発明の第４は、ビール酵母の凝集性を測定する前
記の測定方法に関する。本発明の第５は、測定に使用す
るカラム、ゲル、緩衝液を取りまとめた測定キットに関
する。That is, the first aspect of the present invention relates to a method for measuring the hydrophobicity of the cell surface layer by the adsorption rate of cells to a column packed with a gel equilibrated with a buffer containing a salt. The second aspect of the present invention relates to the above-mentioned measuring method, wherein the cells are yeast and bacteria. A third aspect of the present invention is a pH 4.2 containing salt.
The present invention relates to a method for measuring the degree of hydrophobicity of brewer's yeast by the adsorption rate of brewer's yeast on a column filled with a hydrophobic gel equilibrated with an acetate buffer adjusted as described above. A fourth aspect of the present invention relates to the above-mentioned measuring method for measuring the cohesiveness of brewer's yeast. A fifth aspect of the present invention relates to a measurement kit in which a column, a gel, and a buffer used for measurement are collected.
【０００８】[0008]
【発明の実施の形態】本発明により測定できる細胞は、
酵母、細菌など疎水性を有する細胞であれば何でも測定
することができる。更にその細胞に適した測定条件、た
とえば下面ビール酵母に適した条件を設定することによ
って、その凝集能をとらえることができる。細胞表層の
疎水度の測定条件としては、吸着性の低い材質のカラ
ム、たとえばポリプロピレン製のカラムを用いて、０〜
４Ｍに調整したＮａＣｌ等の塩を含む緩衝液により平衡
化したゲルを充填する。なお、緩衝液やゲルは生体試料
やタンパク質などに広く用いられるものが使用でき、そ
の濃度及び使用する塩、ｐＨ、処理温度は、疎水度を測
定したい細胞や微生物等にダメージがないような範囲で
適宜組み合わせて使用することができる。なお、ビール
の下面酵母について測定する場合は、緩衝液として酵母
細胞にダメージを与えないｐＨ４．２程度に調整したＡ
ｃｅｔａｔｅ Ｂｕｆｆｅｒが好ましく、疎水性のゲル
はフェニルセファロースなどが好ましい。BEST MODE FOR CARRYING OUT THE INVENTION
Any cell can be measured as long as it has a hydrophobic property such as yeast or bacteria. Further, by setting measurement conditions suitable for the cells, for example, conditions suitable for bottom brewer's yeast, the agglutinating ability can be captured. The conditions for measuring the hydrophobicity of the cell surface layer are as follows: a column made of a material having low adsorptivity, for example, a polypropylene column,
The gel equilibrated with a buffer containing a salt such as NaCl adjusted to 4M is filled. Buffers and gels that are widely used for biological samples and proteins can be used, and the concentration, salt, pH, and treatment temperature used are within a range that does not damage cells or microorganisms whose hydrophobicity is to be measured. Can be used in appropriate combination. In addition, when measuring the yeast on the lower surface of beer, A was adjusted to a pH of about 4.2 as a buffer so as not to damage the yeast cells.
Caterate Buffer is preferred, and phenyl sepharose is preferred for the hydrophobic gel.
【０００９】次に、細胞の代謝を抑えダメージを与えな
いよう低温下において、塩を含まない上記緩衝液によ
り、洗浄、懸濁した細胞懸濁液をカラムに供した後、ゲ
ルを平衡化した緩衝液で溶出する。得られた溶出液と、
溶出条件と同倍率の希釈を行った対象の酵母懸濁液につ
いて、ＯＤ ６００〜６６０における吸光度を測定し、
それらの値から細胞のカラムへの吸着率を求め、これを
疎水度と判定する。また、同上の条件でスケールアップ
した疎水性カラムを用い、上記記載の緩衝液及び溶媒で
連続的に溶出することにより、疎水性の異なる細胞を分
取することも可能である。なお、上記測定法に必要なカ
ラム、ゲル、緩衝液等を測定キットとして提供すること
ができる。今回確立した疎水度測定法は、従来法に比べ
て操作が極めて簡単であり、この方法を利用することに
より、下面酵母では凝集能と疎水度の間に確かに相関が
認められ、疎水度を測定することにより容易に凝集性酵
母と非凝集性酵母を区別することができた。Next, the cell suspension washed and suspended with the above salt-free buffer solution was applied to a column at a low temperature so as not to suppress cell metabolism and cause damage, and then the gel was equilibrated. Elute with buffer. The obtained eluate,
The absorbance at OD 600 to 660 was measured for the yeast suspension of the subject subjected to the same dilution as the elution conditions,
From these values, the adsorption rate of the cells to the column is determined, and this is determined as the hydrophobicity. In addition, by using a hydrophobic column scaled up under the same conditions as above and continuously eluting with the above-mentioned buffer solution and solvent, cells having different hydrophobicity can be collected. In addition, a column, a gel, a buffer, and the like necessary for the above-described measurement method can be provided as a measurement kit. The newly established method for measuring the degree of hydrophobicity is much easier to operate than the conventional method. By the measurement, the flocculent yeast and the non-flocculant yeast could be easily distinguished.
【００１０】[0010]
【実施例】以下に実施例を示すが、本発明はこれに限定
されるものではない。 実施例１ ＜試験方法＞非凝集性下面酵母Ａおよび凝集性下面酵母
Ｂを麦汁において１２℃、静置にて１週間発酵させた時
の酵母それぞれを４℃の条件下で、３０００ｒｐｍ、５
分間遠心を行い集菌、１００ｍＭ Ａｃｅｔａｔｅ Ｂ
ｕｆｆｅｒ（ｐＨ４．２）で洗浄、集菌を２回繰り返し
た後、同ｂｕｆｆｅｒにＯＤ６６０における吸光度が約
０．８となるように懸濁して調製した。さらに、酵母懸
濁液Ａ・Ｂを１００：０、７５：２５、５０：５０、２
５：７５、０：１００の割合にて混合した試料を調製
し、以下の試験に供した。EXAMPLES Examples will be shown below, but the present invention is not limited to these examples. Example 1 <Test method> Non-coagulable bottom yeast A and cohesive bottom yeast B were fermented in wort at 12 ° C for 1 week at 4 ° C.
Centrifuge for 100 minutes to collect the cells, 100 mM Acetate B
After repeating washing and collection with a buffer (pH 4.2) twice, the suspension was prepared in the same buffer so that the absorbance at OD660 was about 0.8. Furthermore, yeast suspensions A and B were added at 100: 0, 75:25, 50:50, 2
Samples mixed at a ratio of 5:75 and 0: 100 were prepared and subjected to the following tests.
【００１１】バイオスピンエンプテイカラム（直径０．
６４ｃｍ、高さ５．０ｃｍ；ＢＩＯ−ＲＡＤ社製）に、
１．０Ｍ ＮａＣｌを含む１００ｍＭ Ａｃｅｔａｔｅ
Ｂｕｆｆｅｒ（ｐＨ４．２）により平衡化したゲル、
フェニルセファロースＣＬ−４Ｂ（ファルマシア社製）
０．２５ｍｌを充填し、上記記載方法にて調製した酵母
懸濁液０．１ｍｌを本カラムに供した後、４℃の１．０
Ｍ ＮａＣｌを含む１００ｍＭ Ａｃｅｔａｔｅ Ｂｕ
ｆｆｅｒ（ｐＨ４．２）溶液３ｍｌで溶出した。対象は
酵母懸濁液０．１ｍｌを同溶出ｂｕｆｆｅｒ ３ｍｌに
希釈したものとし、溶出液と対象の希釈液のＯＤ６６０
における吸光度を測定、得られた値から酵母のカラムへ
の吸着率をもとめ、これを疎水度と判定した。なお、非
凝集性株細胞Ａ及び凝集性株細胞Ｂがそれぞれ１００％
の時の疎水度の測定値を基準に、各混合比率での疎水度
を計算し、実測値と比較した。[0011] Biospin empty column (diameter 0. 1)
64 cm, height 5.0 cm; manufactured by BIO-RAD)
100 mM Acetate containing 1.0 M NaCl
Gel equilibrated with Buffer (pH 4.2),
Phenyl Sepharose CL-4B (Pharmacia)
0.25 ml was filled, and 0.1 ml of the yeast suspension prepared by the method described above was applied to the column, and then the mixture was added at 1.0C at 4 ° C.
100 mM Acetate Bu containing M NaCl
The eluate was eluted with 3 ml of an ffer (pH 4.2) solution. The target was prepared by diluting 0.1 ml of the yeast suspension into 3 ml of the same elution buffer.
Was measured and the adsorption rate of the yeast to the column was determined from the obtained value, and this was determined as the hydrophobicity. In addition, the non-aggregating cell line A and the aggregating cell line B were 100% each.
The hydrophobicity at each mixing ratio was calculated based on the measured value of the hydrophobicity at the time of, and compared with the actually measured value.
【００１２】＜試験結果＞本測定法により求めた凝集能
の異なる酵母を混合した懸濁液の疎水度は、酵母の混合
割合にともなって変化し、酵母の各混合比率における実
測値をその計算値と比較した結果、精度良くとらえるこ
とが可能であることが確認できた。<Test Results> The hydrophobicity of a suspension obtained by mixing yeasts having different aggregating abilities determined by the present measurement method changes with the mixing ratio of yeast, and the measured value at each mixing ratio of yeast is calculated. As a result of comparison with the values, it was confirmed that it was possible to capture with high accuracy.
【００１３】[0013]
【発明の効果】本発明によれば、簡易なカラム装置と特
定の緩衝液を利用することにより、細胞表層の疎水性を
従来法よりも簡便な操作で精度良く測定でき、更に下面
ビール酵母表層の疎水性を測定することで、凝集性株へ
の非凝集性株の混合を推定できる疎水度測定法及びその
測定キットを提供することができる。According to the present invention, by using a simple column apparatus and a specific buffer, the hydrophobicity of the cell surface layer can be accurately measured by a simpler operation than the conventional method. By measuring the hydrophobicity of a non-aggregable strain, a method for measuring the degree of hydrophobicity and a kit for measuring the hydrophobicity can be provided.
【図１】 凝集性酵母と非凝集性酵母を混合した系の疎
水度を示すグラフである。FIG. 1 is a graph showing the hydrophobicity of a system in which a flocculating yeast and a non-aggregating yeast are mixed.
Claims (4)
したカラムに対する細胞の吸着率により、細胞表層の疎
水度を測定する方法1. A method for measuring the hydrophobicity of a cell surface layer based on a cell adsorption rate on a column packed with a gel equilibrated with a buffer containing a salt.
測定方法2. The method according to claim 1, wherein the cell is a yeast or a bacterium.
的とする請求項１記載の測定方法3. The method according to claim 1, wherein the cohesiveness of the brewer's yeast is measured.
ム、ゲル、緩衝液を取りまとめた測定キット4. A measurement kit comprising a column, a gel, and a buffer used for the measurement according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12214396A JP2998124B2 (en) | 1996-04-09 | 1996-04-09 | Hydrophobicity measurement method and measurement kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12214396A JP2998124B2 (en) | 1996-04-09 | 1996-04-09 | Hydrophobicity measurement method and measurement kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09271396A JPH09271396A (en) | 1997-10-21 |
| JP2998124B2 true JP2998124B2 (en) | 2000-01-11 |
Family
ID=14828683
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12214396A Expired - Fee Related JP2998124B2 (en) | 1996-04-09 | 1996-04-09 | Hydrophobicity measurement method and measurement kit |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2998124B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6034648B2 (en) * | 2012-10-16 | 2016-11-30 | アサヒビール株式会社 | Method to increase or decrease the hop-derived aroma and taste imparted to beer |
-
1996
- 1996-04-09 JP JP12214396A patent/JP2998124B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH09271396A (en) | 1997-10-21 |
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