JP4148483B2 - Yeast derived protein - Google Patents

Description

【００１２】
カルシウム塩結晶化抑制能は次の様にして評価した。即ち、炭酸水素ナトリウムの２０mＭ水溶液(pＨ８．７)１．５mlに試料水溶液３０μlを添加した後、塩化カルシウムの２０ml水溶液(pＨ８．７)１．５mlを添加し、系の温度を２５℃に維持しながら次式で表される反応をおこない、反応系の波長５７０nmにおける吸光度(Ａ₅₇₀)またはpＨを分光光度計またはpＨ測定計を用いて経時的に測定する:
【化１】

【００１３】
上記の反応系に試料溶液として蒸留水３０μｌを添加したときのＡ₅₇₀の経時変化を図１に示す。吸光度は約２０秒後から急激に増大し、約２００秒後にほぼ最大値に達し、反応はほぼ完了する。また、図１に対するpＨの経時変化を図２に示す。
従って、試料によるカルシウム塩結晶化抑制能(％)は２００秒後のＡ₅₇₀を測定し、次式から算出するのが簡便である:
【数１】

【００１４】
表１から明らかなように、本発明による酵母菌由来タンパク質のカルシウム塩結晶化抑制能(相対活性: １００％)はプロテイナーゼＫを用いる処理によって大幅に低下すると共に、β−ガラクトシダーゼを用いる処理によってかなり低下する。
このことは本発明による酵母菌由来タンパク質が糖タンパク質の１種である可能性を示唆するものである。
【００１５】
上記の精製試料のカルシウム塩結晶化抑制能を１０℃、２０℃、２５℃、３０℃、３５℃、４０℃、５０℃または６０℃で６０分間処理した後で測定し、その結果(熱安定性)を図３に示す。
【００１６】
ケフィアから分離した酵母菌をＹＭ培地(pＨ６．０)中、好気条件下において３０℃で振盪培養させた時の増殖曲線および種々の培養時間後に得られた本発明による酵母菌由来タンパク質のカルシウム塩結晶化抑制能をそれぞれ図４および図５に示す。なお、図４には培地のpＨの経時変化も示す。これらの図の比較から明らかなように、対数増殖期の初期の酵母菌に由来するタンパク質は特に高いカルシウム塩結晶化抑制能を示す。
【００１７】
本発明による酵母菌由来タンパク質は実質的な副作用を伴うことなく、カルシウム塩の結晶化を有効に抑制するので、種々の用途が期待されるが、特に膵臓結石症の処置剤として特に有用である。
従って、本発明の別の観点によれば、前述の酵母菌由来タンパク質を有効成分とする膵臓結石症処置剤が提供される。
【００１８】
本発明による膵臓結石症処置剤は経口投与に適した形態に調製するのが一般的である。このような形態としてはタブレット、トローチ、ドロップ、水性もしくは油性懸濁液、分散性の粉末もしくは顆粒、エマルション、硬質もしくは軟質カプセル、シロップおよびエリキシル等が例示される。これらの経口投与用処置剤は薬剤組成物の製造分野において既知のいずれかの方法によって調製すればよく、また、該処置剤には甘味剤、香味剤、着色剤および防腐剤から選択される１種または２種以上の添加剤を適宜配合してもよい。
【００１９】
なお、タブレットには有効成分と共にタブレットの製造に適した非毒性の賦形剤、例えば、不活性希釈剤(例えば、炭酸ナトリウムやラクトース等)、造粒剤や崩壊剤(例えば、コースターチ、アルギン酸等)、結合剤(例えば、デンプン、ゼラチン等)および潤滑剤(例えば、ステアリン酸マグネシウム、タルク等)等を適宜配合すればよいが、既知の遅延剤(例えば、モノステアリン酸グリセリン、ジステアリン酸グリセリン等)を用いて被覆することによって胃腸管内の崩壊と吸収を遅延させて有効成分が長時間にわたって徐放されるようにしてもよい。
【００２０】
本発明による膵臓結石症処置剤は注射可能な無菌の水性もしくは油性懸濁液の形態に調製してもよい。この種の注射用懸濁液は適当な分散剤もしくは潤滑剤および沈澱防止剤を使用し、既知の方法によって調製すればよい。この場合の懸濁媒体としては、水、リンゲル液、等張性塩化ナトリウム溶液、合成のモノグリセリドやジグリセリドおよびオレイン酸等が例示される。
【００２１】
【実施例】
以下、本発明を実施例によって説明する。
実施例１
酵母エキス３ｇ、麦芽エキス３ｇ、ペプトン５ｇおよびグルコース１０ｇを蒸留水１０００mlに溶解させた培地(pＨ６．０)を用いて３０℃で前培養したケフィアから分離した酵母菌の培養液(ＯＤ₆₆₀＝０．１)１mlを坂口フラスコ(５００ml)に１００mlずつ分注して１２１℃で２０分間高圧滅菌した後で冷却した該培地へ添加し、３０℃で１２０rpmの条件下で２４時間培養した。
菌体を遠心分離し(５０００×ｇ; ４℃; １５分間)、１０mＭトリス塩酸緩衝液(pＨ８．７)を用いて２回処理した。得られた菌体にその約２倍容量の０．５ＭのＥＤＴＡ水溶液(pＨ８．０)を添加した懸濁液を超音波破砕機を用いる４℃での細胞破砕処理に３０秒間隔で１５分間付した後、遠心分離処理(２７７００×ｇ; ４℃; １５分間)に付した。上澄み液を透析チユーブ(「ダイアライシスメンブラン２０」)に入れ、蒸留水を２〜３時間間隔で交換しながら透析処理を一昼夜おこなった。透析物をポリエチレングリコール６０００を用いる濃縮処理に付すことによって酵母菌由来タンパク質を得た。
【００２２】
得られた酵母菌由来タンパク質(濃度: １．５μg／ml、２．１μg／mlまたは６．１μg／ml)のカルシウム塩結晶化抑制能を前述の方法に準拠して調べた。結果を図６(Ａ₅₇₀の経時変化)および図７(pＨの経時変化)に示す。
酵母菌由来タンパク質の濃度をさらに変化させることにより、カルシウム塩の結晶化を完全に抑制する該タンパク質の最低濃度は約３μg／mlであることが判明した。
【００２３】
比較例１
表２に示す成分のカルシウム塩結晶化抑制能を前述の方法に準拠して測定した。結果を表２に示す。
【表２】

【００２４】
表３に示す細菌または酵母を所定の培地で培養した後、実施例１と同様の処理に付し、得られた微生物由来タンパク質(１．４μg／ml)のカルシウム塩結晶化抑制能を前述の方法に準拠して測定した。結果を表３に示す。
【表３】

【００２５】
【発明の効果】
本発明による酵母菌由来タンパク質は食品ケフィアに由来する酵母菌を入手源とするので副作用がないだけでなく、カルシウム塩の結晶化を有効に抑制するので種々の用途が期待されるが、特に膵臓結石症で患う患者の処置剤(治療剤および予防剤)および食品用高カルシウム吸収剤等として有用である。
【図面の簡単な説明】
【図１】 ＮaＨＣＯ₃−ＣaＣl₂反応系における酵母菌由来タンパク質の不存在下でのＡ₅₇₀の経時変化を示すグラフである。
【図２】 ＮaＨＣＯ₃−ＣaＣl₂反応系における酵母菌由来タンパク質の不存在下でのpＨの経時変化を示すグラフである。
【図３】 酵母菌由来タンパク質の処理温度とカルシウム塩結晶化抑制能に関する相対活性との関係を示すグラフである。
【図４】 ケフィアから分離した酵母菌の増殖曲線と培地のpＨの経時変化を示すグラフである。
【図５】 種々の培養時間後に得られた酵母菌由来タンパク質のカルシウム塩結晶化抑制能を示すグラフである。
【図６】 ＮaＨＣＯ₃−ＣaＣl₂反応系における酵母菌由来タンパク質(実施例１で調製した試料)の存在下または不存在下でのＡ₅₇₀の経時変化を示すグラフである。
【図７】 ＮaＨＣＯ₃−ＣaＣl₂反応系における酵母菌由来タンパク質(実施例１で調製した試料)の存在下または不存在下でのpＨの経時変化を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a yeast-derived protein having a calcium salt crystallization inhibitory ability.
[0002]
[Prior art]
Calcium is the most abundant component of the minerals that make up the human body, such as the human body, about 99% of which is a component of bones and teeth, and the remaining about 1% is the activity of various yeasts and the calming of cells. Plays an important role in the life-sustaining action of In the latter case, if a stone formed by crystallization of calcium salt in a specific organ blocks the secretory gland, the living body will die, but in a normal living body, it has a special ability to inhibit calcium salt crystallization. The crystallization is effectively suppressed because of the production of a new protein.
For example, pancreatic juice secreted from the human pancreas into the small intestinal lumen contains supersaturated calcium carbonate in addition to various digestive enzymes, and crystallization of the calcium salt is caused by PSP (Pancreatic Stone) contained in the pancreatic juice. Protein; one of glycophosphoproteins rich in aqueous amino acids) [A. De Caro et al., Biochem. J. et al. 222, 669 (1984)].
However, if this balance breaks down due to some cause (for example, a decrease in the concentration of PSP with aging, etc.), the calcium salt crystallizes and forms a calcite-shaped calculus in the pancreatic duct. Bring.
[0003]
One solution to this problem is to administer PSP ex vivo to patients suffering from such stones, but no method is known for preparing PSP on a practical clinical scale. For this reason, various chelating agents capable of binding to calcite are commercially available as substitutes for PSP, but the fact is that they have not been generally used due to side effects.
[0004]
[Problems to be solved by the invention]
The present invention has been made in view of the above-mentioned actual situation in the field in order to provide a PSP substitute without side effects on a practical clinical scale.
[0005]
[Means for Solving the Problems]
That is, the present invention relates to a yeast-derived protein having calcium salt crystallization inhibitory ability obtained by a preparation method comprising the following steps (i) to (vi):
(i) culturing a yeast isolated from kefir under conditions of a temperature of 5 to 55 ° C. and a pH of 5 to 7,
(ii) treating cultured cells with Tris-HCl buffer,
(iii) subjecting the recovered cells to cell disruption in a state suspended in an EDTA aqueous solution;
(iv) subjecting the supernatant of the cell disrupted product to a nucleic acid removal treatment,
(v) subjecting the supernatant of the nucleic acid removal product to a dialysis treatment;
(vi) The dialyzed product is subjected to a concentration treatment.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The yeast used in the present invention is a yeast of the genus Zygosaccharomyces known per se isolated from kefir. This yeast can be easily separated by conventional separation methods.
[0007]
The culture medium for the yeast isolated from the kefir is not particularly limited, and those commonly used for culturing this type of yeast may be used as appropriate. Examples include 2 to 5 g, malt extract 2 to 5 g, peptone 3 to 8 g and glucose 10 to 15 g.
The pH of the medium is usually adjusted within the range of 5-7. If the pH is out of this range, not only the growth of the fungus will be reduced, but also the ability of the protein finally obtained to suppress crystallization of calcium salt will be unfavorable.
The culture temperature and culture time are not particularly limited, but are usually 5 to 55 ° C, preferably 20 to 40 ° C (particularly 25 to 30 ° C) and 12 to 48 hours (preferably 14 to 36 hours).
Moreover, aerobic conditions are usually employed as the culture atmosphere.
[0008]
The cultured cells are separated by centrifugation. Centrifugation is usually performed at 4000 to 6000 × g at 0 to 5 ° C. for 10 to 20 minutes.
The separated cells are treated once or several times using, for example, 5 to 15 mM Tris-HCl buffer (pH 8.0 to 9.0).
The treated cells are collected by subjecting them to a centrifugation treatment. Centrifugation is usually performed at 25,000 to 30000 × g for 10 to 20 minutes at 0 to 5 ° C.
The collected cells are disrupted by about 0.5 to 3.0 times the volume of cells, for example, suspended in an aqueous solution of 0.3 to 0.6 M EDTA (pH 8.0 to 9.0). Attached. The cell disruption treatment is performed according to a conventional method, for example, using an ultrasonic disrupter or a French press. This cell disruption treatment is usually performed at 0 to 5 ° C. for 10 to 20 minutes.
[0009]
The supernatant obtained by subjecting the cell disrupted product to centrifugation is subjected to nucleic acid removal treatment. Centrifugation in this case is usually performed at 25000 to 30000 × g at 0 to 5 ° C. for 10 to 20 minutes. The nucleic acid removal treatment is carried out at 0 to 4 ° C. according to a conventional method, for example, using streptomycin sulfate and a 0.3 to 0.6 M EDTA aqueous solution (pH 8.0 to 9.0).
The supernatant after the nucleic acid removal treatment is subjected to a dialysis treatment using an appropriate dialysis means. For example, the supernatant is put into a dialysis tube [for example, “Dialysis Membrane 20” (manufactured by WakoChem. USA, Inc.)], and the distilled water is exchanged at intervals of 3 to 4 hours at 0 to 5 ° C. Perform time dialysis.
By concentrating the dialyzate using a suitable concentrating agent such as polyethylene glycol 6000, the yeast-derived protein having the ability to suppress crystallization of calcium salt according to the present invention can be obtained.
[0010]
In order to clarify the characteristics of the yeast-derived protein according to the present invention, the polymer material obtained by the above preparation method is subjected to ultracentrifugation treatment (105,000 × g; 4 ° C .; 2 hours), and then ammonium sulfate. Purification by fractionation (40-80%).
In order to examine the influence of various enzymes on this purified sample, the ability of the purified sample to inhibit crystallization of calcium salt was measured under the conditions of pH 6.0 to 8.0 at 30 to 37 ° C. using various enzymes shown in Table 1. Measurements were made after processing below and the results are shown in Table 1.
[0011]
[Table 1]

[0012]
The ability to suppress calcium salt crystallization was evaluated as follows. That is, 30 μl of a sample aqueous solution was added to 1.5 ml of a 20 mM aqueous solution of sodium bicarbonate (pH 8.7), and then 1.5 ml of a 20 ml aqueous solution of calcium chloride (pH 8.7) was added to maintain the temperature of the system at 25 ° C. Then, the reaction represented by the following formula is carried out, and the absorbance (A ₅₇₀ ) or pH at a wavelength of 570 nm of the reaction system is measured over time using a spectrophotometer or a pH meter:
[Chemical 1]

[0013]
FIG. 1 shows the change over time of A ₅₇₀ when 30 μl of distilled water was added as a sample solution to the above reaction system. Absorbance increases rapidly after about 20 seconds, reaches a maximum value after about 200 seconds, and the reaction is almost complete. Further, FIG. 2 shows a change in pH over time with respect to FIG.
Therefore, the calcium salt crystallization inhibitory ability (%) of the sample can be easily calculated by measuring A ₅₇₀ after 200 seconds and calculating from the following formula:
[Expression 1]

[0014]
As is apparent from Table 1, the ability of the yeast-derived protein according to the present invention to suppress the calcium salt crystallization (relative activity: 100%) is significantly reduced by the treatment with proteinase K, and considerably reduced by the treatment with β-galactosidase. descend.
This suggests the possibility that the yeast-derived protein according to the present invention is a kind of glycoprotein.
[0015]
The above-described purified sample was measured for the calcium salt crystallization inhibiting ability after treatment at 10 ° C., 20 ° C., 25 ° C., 30 ° C., 35 ° C., 40 ° C., 50 ° C. or 60 ° C. for 60 minutes. The property is shown in FIG.
[0016]
Growth curves of yeast isolated from kefir in YM medium (pH 6.0) under aerobic conditions at 30 ° C. and calcium of the protein derived from yeast according to the present invention obtained after various culture times The ability to suppress salt crystallization is shown in FIGS. 4 and 5, respectively. FIG. 4 also shows changes over time in the pH of the medium. As is clear from the comparison of these figures, proteins derived from yeast in the early logarithmic growth phase show particularly high calcium salt crystallization inhibition ability.
[0017]
The yeast-derived protein according to the present invention effectively suppresses the crystallization of calcium salt without substantial side effects, and thus is expected to be used in various ways, but is particularly useful as a treatment for pancreatic stone disease. .
Therefore, according to another aspect of the present invention, there is provided a therapeutic agent for pancreatic stone disease comprising the aforementioned yeast-derived protein as an active ingredient.
[0018]
The treatment for pancreatic stone disease according to the present invention is generally prepared in a form suitable for oral administration. Examples of such forms include tablets, troches, drops, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups and elixirs. These treatment agents for oral administration may be prepared by any method known in the field of manufacturing pharmaceutical compositions, and the treatment agent is selected from sweeteners, flavoring agents, coloring agents and preservatives. You may mix | blend a seed | species or 2 or more types of additives suitably.
[0019]
Tablets include active ingredients and non-toxic excipients suitable for tablet production, such as inert diluents (e.g., sodium carbonate and lactose), granulating agents and disintegrants (e.g., coast starch, alginic acid, etc.). Etc.), binder (e.g. starch, gelatin etc.) and lubricant (e.g. magnesium stearate, talc etc.) etc. may be added as appropriate, but known retarders (e.g. glyceryl monostearate, glyceryl distearate) Etc.), the disintegration and absorption in the gastrointestinal tract may be delayed so that the active ingredient is released gradually over a long period of time.
[0020]
The agent for treating pancreatic stones according to the present invention may be prepared in the form of a sterile injectable aqueous or oily suspension. Such an injectable suspension may be prepared by a known method using an appropriate dispersant or lubricant and an anti-settling agent. Examples of the suspending medium in this case include water, Ringer's solution, isotonic sodium chloride solution, synthetic monoglyceride, diglyceride and oleic acid.
[0021]
【Example】
Hereinafter, the present invention will be described by way of examples.
Example 1
A culture solution of yeast isolated from kefir pre-cultured at 30 ° C. using a medium (pH 6.0) in which 3 g of yeast extract, 3 g of malt extract, 5 g of peptone and 10 g of glucose were dissolved in 1000 ml of distilled water (OD ₆₆₀ = 0 .1) 1 ml was dispensed into Sakaguchi flasks (500 ml) in 100 ml portions, autoclaved at 121 ° C. for 20 minutes, added to the cooled medium, and cultured at 30 ° C. under 120 rpm for 24 hours.
The cells were centrifuged (5000 × g; 4 ° C .; 15 minutes) and treated twice with 10 mM Tris-HCl buffer (pH 8.7). The suspension obtained by adding approximately twice the volume of 0.5 M EDTA aqueous solution (pH 8.0) to the obtained bacterial cells was subjected to cell disruption treatment at 4 ° C. using an ultrasonic disrupter for 15 minutes at 30 second intervals. And then subjected to centrifugation (27700 × g; 4 ° C .; 15 minutes). The supernatant was put into a dialysis tube ("Dialysis Membrane 20"), and the dialysis treatment was carried out all day and night while exchanging distilled water at intervals of 2 to 3 hours. The dialyzate was subjected to a concentration treatment using polyethylene glycol 6000 to obtain a yeast-derived protein.
[0022]
The ability of the obtained yeast-derived protein (concentration: 1.5 μg / ml, 2.1 μg / ml or 6.1 μg / ml) to inhibit crystallization of calcium salt was examined according to the method described above. The results are shown in FIG. 6 (A ₅₇₀ change over time) and FIG. 7 (pH change over time).
It was found that by further changing the concentration of the yeast-derived protein, the minimum concentration of the protein that completely suppresses the crystallization of the calcium salt was about 3 μg / ml.
[0023]
Comparative Example 1
The calcium salt crystallization inhibiting ability of the components shown in Table 2 was measured according to the method described above. The results are shown in Table 2.
[Table 2]

[0024]
After culturing the bacteria or yeast shown in Table 3 in a predetermined medium, it was subjected to the same treatment as in Example 1, and the calcium salt crystallization inhibitory ability of the obtained microorganism-derived protein (1.4 μg / ml) was described above. Measured according to the method. The results are shown in Table 3.
[Table 3]

[0025]
【The invention's effect】
Since the yeast-derived protein according to the present invention is obtained from yeast derived from food kefir, it has no side effects and effectively suppresses crystallization of calcium salts, so various uses are expected. It is useful as a therapeutic agent (therapeutic agent and prophylactic agent) for patients suffering from calculus and a high calcium absorbent for foods.
[Brief description of the drawings]
It is a graph showing temporal change of A ₅₇₀ in the absence of yeast-derived proteins in [1] NaHCO _{3-CaCl 2} reaction system.
FIG. 2 is a graph showing changes in pH over time in the absence of yeast-derived protein in a NaHCO ₃ —CaCl ₂ reaction system.
FIG. 3 is a graph showing the relationship between the processing temperature of a protein derived from yeast and the relative activity relating to the ability to suppress calcium salt crystallization.
FIG. 4 is a graph showing the time course of the growth curve of yeast isolated from kefir and the pH of the medium.
FIG. 5 is a graph showing calcium salt crystallization inhibitory ability of yeast-derived proteins obtained after various culture times.
FIG. 6 is a graph showing the time course of _{A570 in} the presence or absence of a yeast-derived protein (sample prepared in Example 1) in a NaHCO ₃ —CaCl ₂ reaction system.
FIG. 7 is a graph showing the time course of pH in the presence or absence of a yeast-derived protein (sample prepared in Example 1) in a NaHCO ₃ —CaCl ₂ reaction system.

Claims (8)

Yeast-derived protein having calcium salt crystallization inhibitory ability obtained by a preparation method comprising the following steps (i) to (vi):
(i) culturing a yeast isolated from kefir under conditions of a temperature of 5 to 55 ° C. and a pH of 5 to 7,
(ii) treating cultured cells with Tris-HCl buffer,
(iii) subjecting the recovered cells to cell disruption in a state suspended in an EDTA aqueous solution;
(iv) subjecting the supernatant of the cell disrupted product to a nucleic acid removal treatment,
(v) subjecting the supernatant of the nucleic acid removal product to a dialysis treatment;
(vi) The dialyzed product is subjected to a concentration treatment. The protein according to claim 1, wherein 5 to 15 mM Tris-HCl buffer is used. The protein according to claim 1, wherein a 0.3 to 0.6M EDTA aqueous solution is used. The protein according to claim 1, wherein the cell disruption treatment is performed using an ultrasonic disrupter or a French press. The protein according to claim 1, wherein the nucleic acid removal treatment is performed using streptomycin sulfate and an EDTA aqueous solution. The protein according to claim 1, wherein the dialysis treatment is performed using distilled water. The protein according to claim 1, wherein the concentration treatment is performed using polyethylene glycol 6000. An agent for treating pancreatic stone disease comprising the yeast-derived protein according to any one of claims 1 to 7 as an active ingredient.

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