JPH10117794A - Method for producing yeast ribonucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing ribonucleic acid of high purity in a shorter time than conventionally from yeast crushed without denaturing or losing components contained in cells. SOLUTION: Salts are added to a yeast slurry obtained by crushing a suspension containing yeast cells, and heat extraction is performed under alkaline conditions. Ribonucleic acid production method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for producing high-purity ribonucleic acid (hereinafter referred to as RNA
).

[0002]

2. Description of the Related Art Yeast has been widely applied to the fields of food and medicine. In particular, it is rich in proteins, amino acids, and vitamins, including nucleic acid-related substances such as RNA, and is very useful as a health drug and its material (Tomo Itagaki, supervised by Toshiro Goto: Beer yeast useful for health) Story, Sanichi Shobo, 1995). Nucleic acids, which have been attracting attention for diet and rejuvenation needs for several years, have recently been required for prevention and treatment of atopic allergy and adult diseases, as well as for maintaining and improving physical fitness of athletes (199).
5-year version of Healthcare Foods Market Research Volume 1, pp107-119,
Seed Planning Co., Ltd., 1995; Food and Development, Vol. Three
0, No. 2, 22-28 (1994)). Methods for extracting nucleic acids not only from yeast but also from microorganisms include salts such as salt and potassium chloride, acids or alkalis, sodium dodecyl sulfate (SDS)
Chemical methods using a surfactant such as phenol or an extractant such as phenol (JP-A-50-135274, JP-A-51-73192, JP-A-53-73192)
20493, JP-A-52-119483, JP-A-53-20494, JP-A-53-1
24690, JP-A-61-181394, JP-A-62-129219), biochemical methods using lysozyme which is a cell wall lysing enzyme (JP-A-55-92672, JP-A-2-242654), ultrasonic, glass Physical methods of destroying cells using beads, ball mills, high-pressure jet impact, etc. (Japanese Patent Laid-Open Nos. 50-135274, 53-20
493), but they have problems such as insufficient extraction efficiency, thermal or chemical denaturation of cell contents, that is, yeast components, and complicated removal of unnecessary drugs and substances. There are many. In the above-mentioned method using salts, the yeast is heated and stirred in saline solution to extract RNA, and the extracted solution is subjected to ethanol precipitation under acidic conditions to isolate and acquire RNA. If only salt extraction is used, the desalting operation becomes complicated when the salt concentration increases, or RN increases when the heating temperature increases.
Degradation of A progresses, and conversely, if the heating temperature is low, the RNA purity decreases because extraction is incomplete.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for removing unnecessary drugs and substances, which does not provide sufficient RNA extraction efficiency, involves thermal or chemical denaturation of cell contents, that is, yeast components. It solves the problems of conventional methods such as requiring complicated operations such as complicated operations, and provides a method for more efficiently and easily producing highly pure and highly water-soluble RNA from yeast. It is to be.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, the operation of the crushed yeast has been made simpler by extracting the yeast with alkali by heating with salts. To increase extraction efficiency,
Furthermore, they have found that highly pure and highly water-soluble RNA can be produced, and have completed the present invention.

That is, according to the present invention, a salt is added to a yeast slurry obtained by crushing a suspension containing yeast cells, and the suspension is alkaline, preferably pH 9 to 9.5.
It is intended to provide a method for producing ribonucleic acid having a high purity and a very high water solubility, which is obtained by performing the heat extraction in the step (1).

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The yeast used in the present invention can be used without limitation as long as it is edible, such as brewer's yeast and baker's yeast. Examples are Saccharo
myces), Pichia, Hansenu
la), yeast belonging to the genus Candida, and the like. In addition, even in the case of live yeast, heat-treated, chemically treated, biochemically treated, physically treated yeast, etc., it is also possible to use surplus yeast recovered from the brewing process of alcoholic beverages and the like. The method for obtaining the yeast is not particularly limited.

As an example of the present invention, a method for producing RNA using surplus yeast recovered from a beer production process will be described. By crushing the yeast in a slurry state (solid content: about 16%) recovered from the beer production process with a high-pressure jet impact type homogenizer, the yeast cell wall is crushed without denaturing or losing the cell contents, that is, the yeast components, Yeast can be highly atomized. Salts and water are added and mixed to the crushed yeast slurry (solid content: 5% to 10%) thus obtained. As the salts, sodium perchlorate, sodium sulfate, sodium carbonate, ammonium sulfate and the like can be used in addition to chlorides of sodium chloride, potassium chloride and lithium chloride. Among them, sodium chloride is most preferable. The concentration of the sodium chloride solution is 5% to 10
% Is preferable, and 10% is more preferable because the extract is easily centrifuged. The nucleic acid is extracted by subjecting the suspension to a heat treatment at an alkaline pH, preferably pH 9 to 9.5. In this heat treatment, any heat extraction method can be used, and among them, autoclave treatment is desirable. The heating temperature in the autoclave treatment is 90
C. to 110 C., more preferably 100 C., and the heating time is preferably 15 to 60 min.
Minutes are preferred. Proteins are also dissolved in the solution immediately after nucleic acid extraction, and this dissolved protein can be used as a nucleic acid extract after boiling or autoclaving at pH 3.5 and denatured and removed. The nucleic acid extract is precipitated under a sulfuric acid condition of pH 2 or less, and the RNA precipitate is isolated and obtained by centrifugation or filtration. The precipitate is washed with water or precipitated with ethanol, if necessary, and freeze-dried or spray-dried.

[0008]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Beer yeast (Saccharomyce) adjusted to a solid content of 16% concentration
cerevisiae) was crushed once with a high-pressure jet impact homogenizer (Microfluidizer M110-E / H, manufactured by Microfluidics) at a pressure of 1000 kgf / cm ² . 140 g of the crushed yeast solution was thoroughly mixed with 49 ml of water and 21 g of sodium chloride. 2NH
The pH was adjusted to 5, 6, 6.5, 8, 9, and 10 with Cl and pH 4 and 4N NaOH, and each was autoclaved (100 ° C, 30 minutes).
Did. After cooling with water, the supernatant obtained by centrifugation was used as a nucleic acid extract, and the RNA concentrations in the extracts were compared. For analysis of nucleic acid components, Schmidt Tannheiser Schneider (STS)
Method (edited by the Food Analysis Method Committee of the Japan Food Industry Association: Food Analysis Method, pp563-566, Korin Co., 1984; SCHNEIDER, W.
C .: J. Biol. Chem., 164, 747 (1946)). The following nucleic acid component analysis was performed in the same manner. As shown in FIG. 1, the extraction conditions at pH 9 were able to obtain the highest RNA concentration.

Example 2 A brewer's yeast (Saccharomyce) adjusted to a solid content of 16% was prepared.
cerevisiae) was crushed once with a high-pressure jet impact homogenizer (Microfluidizer M110-E / H, manufactured by Microfluidics) at a pressure of 1000 kgf / cm ² . 140 g of the crushed yeast solution was thoroughly mixed with 49 ml of water and 21 g of sodium chloride. 4NN
The pH was adjusted to 9 with aOH, and the extraction efficiencies were compared by autoclave treatment at temperatures of 90, 100, and 110 ° C for 0, 15, 30, 60, and 120 minutes, respectively. As shown in FIG.
The extraction condition at 100 ° C. for 30 minutes was able to obtain the highest RNA concentration.

Example 3 Beer yeast (Saccharomyce) adjusted to a solid content of 16%
s cerevisiae) with a high-pressure jet impact homogenizer (Microfluidizer M110-E / H).
Crushing was performed once at a pressure of 000 kgf / cm ² . 140 g of the crushed yeast solution was thoroughly mixed with 49 ml of water and 21 g of sodium chloride. The pH was adjusted to 9 with 4N NaOH, and the salt concentration was 0,
The extraction efficiencies by autoclave treatment at 6 concentrations of 1, 2.5, 5, 10, and 15% for 30 minutes were compared. As shown in FIG. 3, high RNA was obtained under extraction conditions with a salt concentration of 2.5 to 10%.
Although a concentration could be obtained, 10% sodium chloride was added due to the ease of centrifugation for re-extraction.

Example 4 Beer yeast (Saccharomyce) adjusted to a solid content of 16%
cerevisiae) was crushed once with a high-pressure jet impact homogenizer (Microfluidizer M110-E / H, manufactured by Microfluidics) at a pressure of 1000 kgf / cm ² . 1.4 kg of the crushed yeast solution was thoroughly mixed with 490 ml of water and 210 g of sodium chloride.
Adjust the pH to 9 with 4N NaOH, and autoclave (10
(0 ° C, 30 minutes). After water cooling, the mixture was centrifuged to obtain a supernatant. The precipitate was washed once with water, and each supernatant centrifuged was combined to obtain 800 ml of a nucleic acid extract. Extract 6
The mixture was adjusted to pH 3.5 with NH ₂ SO ₄ , heated at 90 ° C. for 20 minutes, and centrifuged to remove precipitates such as proteins. 10
The supernatant cooled to below ℃ was washed with 1N H ₂ SO ₄ at pH 1.5-2.
The value was adjusted to 0, and RNA was aggregated and precipitated. The RNA precipitate obtained by centrifugation was dissolved in water, twice the amount of ethanol was added, and the mixture was allowed to stand at 4 ° C. overnight. The next day, the mixture was centrifuged, and the precipitate was washed with 70% ethanol and dried by freeze-drying to obtain a white RNA powder.

Table 1 shows the analysis results of the nucleic acid components, loss on drying, total nitrogen and total phosphorus of the four lots.
Table 2 shows the NA powder yield and RNA recovery. In all four lots, very stable products having an RNA purity of 85% or more, a nucleoside / nucleotide content of 5% or less, and a DNA content of 1% or less could be obtained.

[0013]

[Table 1]

[0014]

[Table 2]

Embodiment 5 In this embodiment, a method according to the present invention is applied to a method known in the art (JP-A-53-124690 and JP-B-55-124).
49838). An aqueous suspension of brewer's yeast (Saccharomyces cerevisiae) at a solid content of 16% was sprayed with a high-pressure jet impact homogenizer (Microfluidizer M110-E /
H, Microfluidics)
Crushing was performed once at a pressure of gf / cm ² . 140 g of the crushed yeast solution was thoroughly mixed with 49 ml of water and 21 g of sodium chloride. The pH was adjusted to 9 with 4N NaOH, followed by autoclaving (100 ° C, 30 minutes). After cooling with water, the supernatant obtained by centrifugation was compared with the nucleic acid component of the extract obtained by the conventional method. Table 3 shows the extraction conditions and the amount of the nucleic acid component in the extract in each method. The method using the method of the present invention has the highest RN among the three methods.
A amount could be obtained.

[0016]

[Table 3] Conventional method 1: method described in JP-A-53-124690 Conventional method 2: method described in JP-B-55-49838

Example 6 RNA powder produced by the method of Example 3 and commercially available RNA
The components were compared with the reagents. Table 4 shows the results. The RNA powder obtained according to the present invention, compared to other commercially available RNA reagents,
It had the features of high RNA purity and low nucleoside / nucleotide content.

[0018]

[Table 4]

Example 7 RNA powder produced by the method of Example 3 and commercially available RNA
A solubility comparison test with the reagent was performed. The results of observing the solubility in water, 0.1N HCl solution, 0.1N NaOH solution, pH4 buffer, pH6.8 buffer and pH9 buffer are shown in Table 5.

[0020]

[Table 5]

◎: Melts quickly within 30 seconds just by shaking by hand. ：: Dissolve within 1 minute using a vortex mixer. Δ: Dissolve within 3 minutes using a vortex mixer. X: It does not melt within 3 minutes even when using a vortex mixer. The solubility of the RNA powder obtained by the present invention in neutral to alkaline aqueous solutions was very high as compared with other commercially available RNA reagents.

[0022]

Industrial Applicability The RNA produced by the method of the present invention is white with no yeast odor or yeast taste as in the prior art, is highly pure, has very high water solubility, and is excellent. Also, brewer's yeast (Saccharomyces cerevisiae) may be used as a raw material in the method of the present invention.
In the case of using, since it is surplus yeast generated during the production of beer, the cost not only for yeast but also for yeast cultivation is small, it is very economically advantageous, and the quality for food and the safety are very high. Excellent things can be provided.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of extraction pH on RNA.

FIG. 2 is a graph showing the relationship between RNA concentration, heating temperature, and heating time in RNA extraction.

FIG. 3 is a graph showing the relationship between NaCl concentration and RNA concentration.

Claims (4)

[Claims] 1. A high-purity, water-soluble, salt-added salt is added to a yeast slurry obtained by crushing a suspension containing yeast cells, and the mixture is heated and extracted under alkaline conditions. Very high ribonucleic acid production method. 2. The production method according to claim 1, wherein the crushing treatment is performed by a high pressure jet impact method. 3. The method according to claim 1, wherein the alkalinity is in the range of pH 9 to 9.5. 4. The method according to claim 1, wherein the yeast is brewer's yeast.