CN108220280B - Method for extracting high-purity nucleic acid by using yeast, product and application thereof - Google Patents
Method for extracting high-purity nucleic acid by using yeast, product and application thereof Download PDFInfo
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- CN108220280B CN108220280B CN201611118650.1A CN201611118650A CN108220280B CN 108220280 B CN108220280 B CN 108220280B CN 201611118650 A CN201611118650 A CN 201611118650A CN 108220280 B CN108220280 B CN 108220280B
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- 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
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
Abstract
The invention relates to the field of nucleic acid extraction, and particularly relates to a method for extracting high-purity nucleic acid by using yeast, a product and application thereof. The method for extracting high-purity nucleic acid by using yeast comprises the following steps: (1) heat extraction: preparing a raw material containing yeast into a solution, and carrying out hot extraction; (2) enzymolysis: and (2) sequentially adding alkaline protease and exoprotease into the solution subjected to heat extraction in the step (1) for enzymolysis. The invention also comprises the nucleic acid prepared by the method and application of the nucleic acid in the fields of food, feed or pharmacy. The nucleic acid in the yeast is extracted by adopting a heat extraction and special double enzymolysis method, the extraction production period is short, and the production process is simple. Is suitable for industrial mass production. The prepared nucleic acid has high purity. Solves the problems of low extraction rate of yeast nucleic acid and low product purity.
Description
Technical Field
The invention relates to the field of nucleic acid extraction, and particularly relates to a method for extracting high-purity nucleic acid by using yeast, a product and application thereof.
Background
At present, many researches on extracting nucleic acid from yeast are carried out, but the application is not many, and no reports of industrial application exist, and different methods have certain limitations: the high-pressure homogenization method has high mechanical strength, the crushing operation is easy to be damaged by shearing, and the method has no selectivity and is easy to completely damage cell walls, thereby increasing the exudation of other impurities and bringing troubles to the purification of nucleic acid; the conditions of the autolysis method are difficult to control, the time consumption is long, and the extraction rate is low; the enzyme dissolution method often has the problem of inhibition, the product inhibition can cause the release rate of intracellular substances to be reduced, meanwhile, the enzyme method has higher wall breaking cost, and the effect is difficult to obtain by single enzyme; although the extraction rate of nucleic acid extracted by the alkaline method is relatively high, more protein is easily mixed to reduce the purity of the nucleic acid, and the post-treatment process is increased, so that the method is to be further improved; the salt method has the advantages of mild extraction conditions, low cost and the like, but the salt method still has a plurality of defects due to high salt content, and the extraction rate is low.
The Chinese patent of invention discloses a file with application number of 94115437.8, named as a production process for extracting nucleic acid from natural plant cells, which takes defatted soybean meal as a raw material and prepares nucleic acid extract and nucleic acid purified dry products through the production processes of swelling, homogenate preparation, cell lysis, solvent extraction, dialysis treatment, concentration and purity determination, sterilization, split charging, drying and the like, and has the following defects: the extraction is carried out by using chemical reagents such as phenol or ethanol, the extract needs to be dialyzed, generally 48 to 72 hours, and the dialysate is replaced every 6 to 8 hours, so that the production period is long, the cost is high, the discharge of harmful liquid can pollute the environment, and the method is not suitable for industrial production.
The Chinese invention patent also discloses a document with the application number of 931110416, namely a preparation method and application of human placenta nucleic acid, the method also utilizes chemical reagents such as saturated phenol for extraction, the dialysis treatment of extract usually needs 5 to 7 days, the production period is long, the cost is high, the discharge of harmful liquid can pollute the environment, the raw material source is less, and the method is not suitable for industrial large-scale production.
Chinese patent No. CN96115417, entitled method for preparing nucleic acid raw material from natural food, is a method for preparing nucleic acid from raw materials of beans, sorghum, barley, wheat and corn fruits, including the process steps of raw material grinding, homogenization cracking and centrifugation to prepare nucleic acid, and is characterized by that it adopts the physical methods of grinding, high-temperature, high-speed homogenization and the like, and makes the cell and nucleoprotein cracking and physical and chemical cutting of nucleic acid macromolecule under the alkaline state using sodium chloride solution as solvent. The invention still uses non-edible raw materials such as ammonium sulfate, and the waste liquid discharge still can not be used for food. The raw materials used by the invention are agricultural and sideline products, the quality fluctuation is large, and the quality of the industrialized mass production is not easy to keep stable.
Disclosure of Invention
The problems of the prior art solved by the invention are as follows: the existing methods for extracting nucleic acid from yeast have certain limitations, and are not suitable for industrial production. When the existing method for extracting nucleic acid by enzymolysis is used, the extraction efficiency of enzyme is not high, a single enzyme cannot obtain a good extraction effect, and then high nucleic acid cannot be extracted.
Therefore, it is desirable to provide a method for extracting nucleic acid with high purity using yeast.
The object of the present invention is to provide a method for extracting nucleic acid with high purity from yeast. Simple process, short production period, small quality fluctuation of raw materials and easy large-scale industrial production.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides a method for extracting high purity nucleic acid using yeast, comprising the steps of:
(1) heat extraction: preparing a raw material containing yeast into a solution, and carrying out hot extraction;
(2) enzymolysis: and (2) sequentially adding alkaline protease and exoprotease into the solution subjected to heat extraction in the step (1) for enzymolysis.
Preferably, the temperature of the heat extraction in step (1) is 90-100 ℃.
Preferably, the temperature of the heat extraction in step (1) is 96-100 ℃.
Preferably, the pH value of the solution in the heat extraction in the step (1) is 7.0-8.0, and the heat extraction time is 60-120 minutes.
Preferably, the weight proportion of the yeast-containing raw material in the solution in the step (1) is 10-30%.
Preferably, the weight proportion of the yeast-containing raw material in the solution in the step (1) is 10-15%.
Preferably, the alkaline protease comprises: novo protease and Carsberg protease.
Preferably, the alkaline protease is derived from Bacillus subtilis.
Preferably, the weight ratio of the alkaline protease in the step (2) to the yeast-containing raw material is 1-5%, preferably 1-3%.
Preferably, the amount of the alkaline protease in the step (2) is 20U to 100U per 1kg of the yeast-containing raw material.
Preferably, the enzymolysis temperature of the alkaline protease is 60-70 ℃, the pH value is 7.0-8.0, and the enzymolysis time is 3-6 h.
Preferably, the exoprotease includes an aminopeptidase and a carboxypeptidase, preferably an aminopeptidase.
Preferably, in the step (2), the concentration of the exoprotease in the raw material containing yeast is 1-5 per thousand, preferably 1-2 per thousand, and more preferably, the amount of the exoprotease is 20U-100U per 1kg of the raw material containing yeast.
Preferably, in step (2), the amount of exoprotease is 0.2U to 1U per 1kg of yeast-containing raw material.
Preferably, the enzymolysis temperature of the exoprotease is 60-70 ℃, the pH value is 7.0-8.0, and the enzymolysis time is 3-6 h.
Preferably, the method further comprises: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
Preferably, the nucleic acid extract is filtered, preferably by membrane filtration, to obtain the extracted nucleic acid solution.
Preferably, the yeast-containing material contains 20 wt% or more of nucleic acid with respect to 100 wt% of baker's yeast cells, wherein the RNA content is more than 12 wt%
In a second aspect, the invention provides a nucleic acid prepared by the method of any one of the above.
In a third aspect, the invention provides the use of a nucleic acid as described above in the food, feed or pharmaceutical field.
The present invention has the following advantages compared to the prior art.
(1) The nucleic acid in the yeast is extracted by adopting a heat extraction and special double enzymolysis method, the extraction production period is short, and the production process is simple. Is suitable for industrial mass production.
(2) In the whole production, the nucleic acid product and the by-product after extracting nucleic acid can be used in food.
(3) The prepared nucleic acid has high purity. Solves the problems of low extraction rate of yeast nucleic acid and low product purity.
Detailed Description
As described above, the present invention provides a method for extracting a high purity nucleic acid using a yeast-containing raw material. The high-purity nucleic acid means that the purity of ribonucleic acid is more than 80%.
Based on the existing enzymatic method for extracting nucleic acid, the invention adopts two enzymes with different properties for enzymolysis in sequence on the basis of heat extraction, namely, the two enzymes are respectively subjected to enzymolysis through alkaline protease and exoprotease, so that the purity of the nucleic acid can be obviously improved.
Alkaline proteases are enzymes which hydrolyze peptide bonds of proteins in the alkaline range of pH, have a pH of 7 to 11 and are also called serine proteases because their active center contains Ser. Not only can hydrolyze peptide bonds, but also has the functions of hydrolyzing ester bonds, amido bonds, transesterification and transpeptidation. Can decompose the peptide chain of macromolecular protein molecules into polypeptide or amino acid, and has stronger capability of decomposing protein. The molecular weight of the alkaline protease is preferably 26000-34000, and the pH is preferably 7-8. Two common alkaline proteases are available, one being the Novo protease and the other being the Carsberg protease. The main source of alkaline protease is microbial extraction, wherein in a preferred embodiment the alkaline protease is from bacillus subtilis. There are several main groups, such as Subtilisin E, Subtilisin BPN, Subtilisin, Carsberg, Subtilisin, and Armylosaccharticus.
Exoproteases are capable of cleaving a single amino acid from either end of a peptide chain, thereby breaking down a protein or polypeptide into individual amino acids. Including aminopeptidases and carboxypeptidases. The present invention is preferably an aminopeptidase.
The method is realized by the following steps:
1. raw material yeast heat extraction
Adding water into yeast-containing raw material, adjusting dry matter concentration to 10-15%, adjusting pH to 7.0-8.0, heating to 90-100 deg.C, and maintaining for 60-120 min.
2. Protein degradation in nucleic acids by addition of enzyme preparations
Adding alkaline protease 1-3 ‰, setting temperature at 60-70 deg.C, pH7.0-8.0, and performing enzymolysis for 3-6 hr, adding exoprotease 1-2 ‰, setting temperature at 60-70 deg.C, pH7.0-8.0, and performing enzymolysis for 3-6 hr. Separating and taking supernatant to obtain nucleic acid extracting solution.
3. Membrane filtration purification of nucleic acids
Filtering the nucleic acid extracting solution by using an organic ultrafiltration membrane, wherein the part intercepted by the membrane is the purified nucleic acid solution.
4. Extraction of nucleic acids
Adding hydrochloric acid into the supernatant, adjusting pH to 2.0-2.5, and precipitating nucleic acid to obtain nucleic acid with high purity.
Among them, the heat treatment is not particularly limited, and the main purpose thereof is to inactivate nucleases (particularly nucleases), proteases, phosphate synthetases and the like in yeast, and the temperature of action thereof is 90 to 100 ℃, preferably 96 to 100 ℃. Too low a temperature may not sufficiently inactivate the enzyme, resulting in a lower content and lower purity of nucleic acid. When the temperature is too high, the product may have a scorched odor or develop color.
Wherein, in the enzyme treatment process, the cell wall of the yeast is firstly broken by alkaline protease, so that nucleic acid in the cell overflows, and the protease removes and degrades protein bound on the nucleic acid. If the treatment is carried out by first using exoprotease and then using alkaline protease, the effect of breaking the yeast cell wall is poor, and much nucleic acid remains in the cell and cannot be collected. Thereby reducing the extraction rate.
Wherein the yeast-containing material is any yeast-containing material. Among them, the "yeast" of the present invention is not particularly limited, and may be, for example, brewers yeast such as Saccharomyces cerevisiae, and also yeast of other yeast strains, Candida, Pichia, Hansenula, and the like. The yeast may be fresh yeast or active dry yeast. It may be a yeast extract or yeast fermentation product or yeast extract containing nucleic acid, or a baker's yeast fermented from molasses or starch hydrolyzing sugar. The baker's yeast can be any commercially available active dry yeast for common noodles, any active dry yeast for small package noodles sold in supermarkets or convenience stores or fresh yeast squeezed into blocks.
In a preferred embodiment of the present invention, the baker's yeast is a high nucleic acid baker's yeast, and the nucleic acid is contained in an amount of 20 wt% or more based on 100 wt% of baker's yeast cells, and the RNA in the nucleic acid is more than 12.0 wt%. The preparation method of the high nucleic acid baker's yeast comprises the seeding tank culture of baker's yeast thalli and the expansion culture of baker's yeast thalli, and comprises the following steps:
a) in the process of the expanded culture, after the wet weight of the baker's yeast reaches 100 g/L-120 g/L, adding glutamine of which the weight is more than 1 per thousand g/L and aspartic acid of which the weight is more than 1 per thousand g/L relative to a fermentation system of the expanded culture;
b) the scale-up culture was continued until the RNA content was more than 12.0 wt% based on 100 wt% of the baker's yeast cells. The preparation method of the bread yeast with high nucleic acid is recorded in Chinese patent ZL 201010613463.7.
The present invention will be described in further detail with reference to specific embodiments. However, these examples are merely representative of the present invention and should not be construed as limiting the scope of the practice of the present invention. In addition, all simple substitutions or changes of the formula and the production process steps of the invention belong to the protection scope of the invention. The starting materials used in the compositions of the invention are those commonly used by those skilled in the art.
Wherein, the raw material information and the manufacturer used in the examples and comparative examples of the present invention are as shown in Table 1:
TABLE 1 raw material information and manufacturer used in examples and comparative examples
| Raw materials | Purity and/or type | Manufacturer of the product |
| Bread yeast | -- | Angel Yeast Co Ltd |
| Alcalase enzyme | 20 ten thousand U/g | Denmark Novoverin Co Ltd |
| Flavourzyme enzyme | 2000U/g | Denmark Novoverin Co Ltd |
| Nuclease enzymes | 50 ten thousand U/g | Nanjing Pombo bioengineering Co.Ltd |
| Papain | 60 ten thousand U/g | Nanjing Pombo bioengineering Co.Ltd |
| Organic ultrafiltration membrane | UFX5，5KD | Alfa Lava Inc. (Afa Laval) |
Example one
100kg of bread dry yeast is taken and added with 1000kg of water to prepare yeast emulsion. Adjusting pH value to 7.5 with sodium hydroxide, heating to 96 deg.C, keeping the temperature for 90 min, cooling to 65 deg.C, adding Alcalase 0.2kg, adjusting pH to 7.0, keeping the temperature at 65 deg.C, performing enzymolysis for 4 hr, and inactivating; then, 0.15kg of flavourazyme enzyme was added, the temperature was maintained at 60 ℃ and the pH was 7.0, and after 4 hours of action, inactivation was carried out. Then, the mixture was centrifuged at 5000 rpm for 30 minutes in a centrifuge to obtain a supernatant. Filtering the supernatant with organic ultrafiltration membrane (molecular weight 5KD), and collecting the liquid as nucleic acid extractive solution. Adding hydrochloric acid into the nucleic acid extract, adjusting pH to 2-2.5, and collecting precipitate to obtain nucleic acid. Washing the obtained nucleic acid precipitate with clear water to obtain high-purity nucleic acid. Then, freeze-drying was carried out to obtain a nucleic acid powder.
Then, the content of nucleic acid after extraction and the content of nucleic acid in yeast before extraction were measured by the following methods.
The specific determination method is as follows:
the method for measuring the content of the nucleic acid by referring to the RNA content in annex A in the enterprise standard Q/AQJM 2144S-2014 of Angel Yeast GmbH comprises the following steps:
1. reagent
0.5mol/L perchloric acid (HClO)4) Solution: 21.5mL 70% (or 22.1mL 68%) perchloric acid was added to 400mL of distilled water, and the volume was made 500mL with distilled water.
0.25mol/L perchloric acid (HClO)4) Solution: taking 250mL0.5mol/L perchloric acid (HClO)4) Diluting with distilled water to 500 mL.
2. Device
An electronic balance: accurate to 1 mg;
a centrifuge: 4000 revolutions per minute, yeast can be separated;
centrifuging the tube to a capacity of at least 10 mL;
a spectrophotometer: the wavelength can be adjusted to 260 nm;
hot water bath: 70 ℃;
cooling water bath: 4 ℃;
10mL and 1mL pipettes;
100mL volumetric flask.
3. Procedure for the preparation of the
3.1 sample treatment
Pipette 20-50mg of sample into the tube and weigh it.
5mL of 0.5mol/L HClO was added4And oscillating and mixing uniformly. The centrifuge tube is placed in a 70 ℃ water bath kettle, the temperature is kept for 15 minutes, and the centrifuge tube is oscillated once every 3 to 4 minutes.
After centrifugation at 4000rpm for 10 minutes, 1mL of the supernatant was aspirated, dissolved in distilled water to 100mL, and mixed well.
3.2 determination of
The cuvette is rinsed with the sample to be tested and filled into the spectrophotometer. The surface is wiped clean. The absorbance was measured at 260nm using distilled water as a blank. The absorbance was recorded and the measurement was repeated once, and the average of the two measurements was taken.
Computing
RNA content (%) - (a × dilution × 0.03365 × 5 × 100) ÷ (m × Ds)
Wherein A is the absorbance of the sample solution;
dilution-when operating according to the method, the dilution is 100;
m-weight of sample, mg;
Ds-Dry matter of sample;
5-adding 0.5mol/LHClO4The volume of post-solution;
0.03365-corresponding to the absorbance of 1.00, the RNA content in the solution to be tested, mg/100 mL.
4. Precision degree
The absolute difference between two independent measurements obtained under repetitive conditions must not exceed 3% of the arithmetic mean.
The measured content of nucleic acid after extraction and the value of nucleic acid content in yeast before extraction were recorded separately, and then the extraction rate of nucleic acid and the purity of nucleic acid were calculated.
Wherein the extraction rate of nucleic acid is the percentage of the content of nucleic acid in yeast measured after extraction.
The purity of the nucleic acid is the content of the nucleic acid measured after extraction as a percentage by weight of the nucleic acid after freeze-drying.
The extraction rate of nucleic acid calculated in the first example was 72% and the purity of nucleic acid was 85%.
Meanwhile, the sensory, physicochemical indexes and the microbial limit of the prepared nucleic acid are determined by referring to the enterprise standard Q/AQJM 2144S-2014 of Angel Yeast GmbH, and all the indexes meet the requirements.
Wherein the water content: measured according to the method specified in GB 5009.3.
Total arsenic: measured according to the method specified in GB/T5009.11.
Lead: measured according to the method specified in GB 5009.12.
Total number of colonies: measured according to the method specified in GB 4789.2.
Coliform group bacteria: measured according to the method specified in GB 4789.3.
And (3) mould: measured according to the method specified in GB 4789.15.
Salmonella: measured according to the method specified in GB 4789.4.
TABLE 2 organoleptic characteristics
| Item | Feature(s) |
| Color | Light yellow. |
| Smell(s) | Nucleic acids have a characteristic odor. |
| Appearance of the product | And (3) powder. |
| Impurities | No foreign matter is visible to the naked eye. |
TABLE 3 physical and chemical indexes
| Item | Parameter(s) |
| RNA content (on a dry basis),% of | ≥75.0 |
| Water content% | ≤8.0 |
| The total arsenic (in terms of As),mg/kg | ≤2.0 |
| lead (in terms of Pb), mg/kg | ≤2.0 |
TABLE 4 microbial limits
| Item | Parameter(s) |
| Total number of colonies, CFU/g | ≤10000 |
| Coliform group, MPN/g | ≤0.3 |
| Mold, CFU/g | ≤25 |
| Yeast, CFU/g | ≤25 |
| Salmonella,/25 g | Not detected out |
Example two
100kg of bread dry yeast is taken and added with 900kg of water to prepare yeast emulsion. Adjusting pH value to 8 with sodium hydroxide, heating to 95 deg.C, keeping temperature for 120 min, cooling to 70 deg.C, adding Alcalase 0.1kg, adjusting pH to 7.5, maintaining temperature at 70 deg.C, performing enzymolysis for 3 hr, and inactivating; then, 0.1kg of flavourazyme enzyme was added, the temperature was maintained at 70 ℃ and the pH was 7.5, and after 3 hours of action, inactivation was carried out. Then, the mixture was centrifuged at 5000 rpm for 30 minutes in a centrifuge to obtain a supernatant. Filtering the supernatant with organic ultrafiltration membrane (molecular weight 5KD), and collecting the liquid as nucleic acid extractive solution. Adding hydrochloric acid into the nucleic acid extract, adjusting pH to 2-2.5, and collecting precipitate to obtain nucleic acid. Washing the obtained nucleic acid precipitate with clear water to obtain high-purity nucleic acid. Then, the mixture was cooled and dried to obtain a nucleic acid powder.
Then, the extraction rate of nucleic acid and the purity of nucleic acid were calculated according to the method of example one.
The extraction rate of nucleic acid is 71%, and the purity of nucleic acid is 80%. The sensory and physicochemical indexes of the prepared nucleic acid and the indexes of the limited amount of the microorganism all meet the requirements.
EXAMPLE III
100kg of bread dry yeast is taken and added with 600kg of water to prepare yeast emulsion. Adjusting pH to 7.0 with sodium hydroxide, heating to 100 deg.C, keeping the temperature for 60 min, cooling to 65 deg.C, adding Alcalase 0.3kg, adjusting pH to 8.0, keeping the temperature at 60 deg.C, performing enzymolysis for 6 hr, and inactivating; then 0.2kg of flavourazyme enzyme is added, the temperature is kept at 60 ℃, the pH value is 8.0, and after 6 hours of action, the enzyme is inactivated. Then, the mixture was centrifuged at 5000 rpm for 30 minutes in a centrifuge to obtain a supernatant. Filtering the supernatant with organic ultrafiltration membrane (molecular weight 5KD), and collecting the liquid as nucleic acid extractive solution. Adding hydrochloric acid into the nucleic acid extract, adjusting pH to 2-2.5, and collecting precipitate to obtain nucleic acid. Washing the obtained nucleic acid precipitate with clear water to obtain high-purity nucleic acid. Then, the mixture was cooled and dried to obtain a nucleic acid powder.
Then, the extraction rate of nucleic acid and the purity of nucleic acid were calculated according to the method of example one. The extraction rate of nucleic acid was 74% and the purity of nucleic acid was 90% by experiment. The sensory and physicochemical indexes of the prepared nucleic acid and the indexes of the limited amount of the microorganism all meet the requirements.
Comparative example 1
100kg of bread dry yeast is taken and added with 900kg of water to prepare yeast emulsion. Adjusting pH to 8.0 with sodium hydroxide, heating to 85 deg.C, maintaining the temperature for 150 min, cooling to 70 deg.C, adding Alcalase 0.1kg, adjusting pH to 7.5, maintaining the temperature at 70 deg.C, performing enzymolysis for 3 hr, and inactivating; then, 0.1kg of flavourazyme enzyme was added, the temperature was maintained at 70 ℃ and the pH was 7.5, and after 3 hours of action, inactivation was carried out. Then, the mixture was centrifuged at 5000 rpm for 30 minutes in a centrifuge to obtain a supernatant. Filtering the supernatant with organic ultrafiltration membrane (molecular weight 5KD), and collecting the liquid as nucleic acid extractive solution. Adding hydrochloric acid into the nucleic acid extract, adjusting pH to 2-2.5, and collecting precipitate to obtain nucleic acid. Washing the obtained nucleic acid precipitate with clear water to obtain nucleic acid solution. Then, the mixture was cooled and dried to obtain a nucleic acid powder.
Then, the extraction rate of nucleic acid and the purity of nucleic acid were calculated according to the method of example one.
The extraction rate of the nucleic acid is 60% and the purity of the nucleic acid is 80% according to the experiment. The sensory and physicochemical indexes of the prepared nucleic acid and the indexes of the limited amount of the microorganism all meet the requirements.
Comparative example No. two
100kg of bread dry yeast is taken and added with 900kg of water to prepare yeast emulsion. Adjusting pH value to 8.0 with sodium hydroxide, heating to 95 deg.C, maintaining temperature for 120 min, cooling to 70 deg.C, adding Amano nuclease 0.1kg, adjusting pH to 7.5, maintaining temperature at 70 deg.C, performing enzymolysis for 3 hr, and inactivating; then adding papain 0.1kg, keeping the temperature at 70 deg.C and pH at 7.5, acting for 3 hr, and inactivating. Then, the mixture was centrifuged at 5000 rpm for 30 minutes in a centrifuge to obtain a supernatant. Filtering the supernatant with organic ultrafiltration membrane (molecular weight 5KD), and collecting the liquid as nucleic acid extractive solution. Adding hydrochloric acid into the nucleic acid extract, adjusting pH to 2-2.5, and collecting precipitate to obtain nucleic acid. Washing the obtained nucleic acid precipitate with clear water to obtain nucleic acid solution. Then, the mixture was cooled and dried to obtain a nucleic acid powder.
Then, the extraction rate of nucleic acid and the purity of nucleic acid were measured by the method of example one.
The extraction rate of nucleic acid is only 20% and the purity of nucleic acid is 57%. Since the enzymolysis was performed using Amano nuclease, the extracted nucleic acid was decomposed into nucleotides, and the extraction rate of nucleic acid was only 20%. But also loses the extraction significance because most of the nucleic acid is degraded.
Comparative example No. three
100kg of bread dry yeast is taken and added with 900kg of water to prepare yeast emulsion. Adjusting pH to 8.0 with sodium hydroxide, heating to 95 deg.C, maintaining for 120 min, cooling to 65 deg.C, adding flavoenzyme 0.1kg, adjusting pH to 7.0, maintaining temperature at 65 deg.C, performing enzymolysis for 3 hr, and inactivating; then 0.1kg of Alcalase enzyme is added, the temperature is kept at 70 ℃, the pH value is 7.0, and after 3 hours of action, the Alcalase enzyme is inactivated. Then, the mixture was centrifuged at 5000 rpm for 30 minutes in a centrifuge to obtain a supernatant. Filtering the supernatant with organic ultrafiltration membrane (molecular weight 5KD), and collecting the liquid as nucleic acid extractive solution. Adding hydrochloric acid into the nucleic acid extract, adjusting pH to 2-2.5, and collecting precipitate to obtain nucleic acid. Washing the obtained nucleic acid precipitate with clear water to obtain nucleic acid solution. Then, the mixture was cooled and dried to obtain a nucleic acid powder.
Then, the extraction rate of nucleic acid and the purity of nucleic acid were measured by the method of example one.
The extraction rate of nucleic acid is 50% and the purity of nucleic acid is 60%. In the third comparative example, firstly, exonuclease flavourozyme is used for enzymolysis, and then alkaline protease Alcalase is used for enzymolysis, the effect after the treatment is the same as that of the first to third examples, and both the extraction rate of nucleic acid and the purity of nucleic acid are lower. The two protease proteases are exchanged in sequence, namely alkaline protease is not utilized for enzymolysis firstly, the breaking effect on yeast cell walls is poor, so that a lot of nucleic acid is remained in cells, is wasted along with the centrifugal process and cannot be collected, and the extraction rate of the nucleic acid is reduced. And the purity of nucleic acid is low due to poor enzymolysis effect.
Comparative example No. four
100kg of bread dry yeast is taken and added with 900kg of water to prepare yeast emulsion. Adjusting pH to 8.0 with sodium hydroxide, heating to 95 deg.C, maintaining the temperature for 120 min, cooling to 70 deg.C, adding papain 0.1kg, maintaining the temperature at 70 deg.C and pH at 7.5, acting for 6 hr, and inactivating. Then, the mixture was centrifuged at 5000 rpm for 30 minutes in a centrifuge to obtain a supernatant. Filtering the supernatant with organic ultrafiltration membrane (molecular weight 5KD), and collecting the liquid as nucleic acid extractive solution. Adding hydrochloric acid into the nucleic acid extract, adjusting pH to 2-2.5, and collecting precipitate to obtain nucleic acid. Washing the obtained nucleic acid precipitate with clear water to obtain nucleic acid solution. Then, the mixture was cooled and dried to obtain a nucleic acid powder.
Then, the extraction rate of nucleic acid and the purity of nucleic acid were measured by the method of example one.
The extraction rate of the nucleic acid is 40% and the purity of the nucleic acid is 55% according to the experiment. Only single enzyme (papain) is adopted for enzymolysis, the enzymolysis effect is poor, the measured extraction rate of nucleic acid and the purity of the nucleic acid are both low, and the purpose of application cannot be achieved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and all such modifications, equivalents and improvements that come within the spirit and scope of the invention are desired to be protected.
Claims (60)
1. A method for extracting high-purity nucleic acid by using yeast is characterized by comprising the following steps:
(1) heat extraction: preparing a raw material containing yeast into a solution, and carrying out hot extraction;
(2) enzymolysis: sequentially adding alkaline protease and exoprotease into the solution subjected to heat extraction in the step (1) for enzymolysis;
the temperature of the heat extraction in the step (1) is 96-100 ℃.
2. The method according to claim 1, wherein the solution in the heat extraction in the step (1) has a pH of 7.0 to 8.0 and the heat extraction time is 60 to 120 minutes.
3. The method according to claim 1, wherein the yeast-containing material is present in the solution in an amount of 10 to 30% by weight in step (1).
4. The method according to claim 2, wherein the yeast-containing material is present in the solution in an amount of 10 to 30% by weight in step (1).
5. The method according to claim 1, wherein the yeast-containing material is present in the solution in a proportion of 10 to 15% by weight in step (1).
6. The method according to claim 2, wherein the yeast-containing material is present in the solution in a proportion of 10 to 15% by weight in step (1).
7. The method of claim 1, wherein the alkaline protease comprises: novo protease and Carsberg protease.
8. The method according to claim 2, wherein the alkaline protease comprises: novo protease and Carsberg protease.
9. The method according to claim 3, wherein the alkaline protease comprises: novo protease and Carsberg protease.
10. The method according to claim 4, wherein the alkaline protease comprises: novo protease and Carsberg protease.
11. The method of claim 5, wherein the alkaline protease comprises: novo protease and Carsberg protease.
12. The method of claim 6, wherein the alkaline protease comprises: novo protease and Carsberg protease.
13. The method according to any one of claims 7 to 12, wherein the alkaline protease is derived from bacillus subtilis.
14. The method according to any one of claims 1 to 12, wherein the weight ratio of the alkaline protease to the yeast-containing raw material in step (2) is 1 to 5% o.
15. The method according to any one of claims 1 to 12, wherein the weight ratio of the alkaline protease to the yeast-containing raw material in step (2) is 1 to 3% o.
16. The method according to any one of claims 1 to 12, wherein the amount of the alkaline protease is 20U to 100U per 1kg of the yeast-containing raw material.
17. The method according to any one of claims 1 to 12, wherein the temperature of the alkaline protease is 60 to 70 ℃, the pH value is 7.0 to 8.0, and the enzymolysis time is 3 to 6 hours.
18. The method of claim 14, wherein the temperature of the alkaline protease is 60-70 ℃, the pH value is 7.0-8.0, and the enzymolysis time is 3-6 h.
19. The method of claim 16, wherein the temperature of the alkaline protease is 60-70 ℃, the pH value is 7.0-8.0, and the enzymolysis time is 3-6 h.
20. The method of any one of claims 1-12, wherein the exoprotease comprises an aminopeptidase and/or carboxypeptidase.
21. The method of claim 14, wherein the exoprotease comprises an aminopeptidase and/or carboxypeptidase.
22. The method of claim 16, wherein the exoprotease comprises an aminopeptidase and/or carboxypeptidase.
23. The method of claim 17, wherein the exoprotease comprises an aminopeptidase and/or carboxypeptidase.
24. The method of any one of claims 1 to 12, wherein the exoprotease is an aminopeptidase.
25. The method according to any one of claims 1 to 12, wherein the concentration of exoprotease in step (2) is 1 to 5% o by weight of the yeast-containing raw material.
26. The method according to claim 14, wherein the concentration of exoprotease in step (2) is 1-5% o by weight of the yeast-containing material.
27. The method according to claim 16, wherein the concentration of exoprotease in step (2) is 1-5% o by weight of the yeast-containing material.
28. The method according to claim 17, wherein the concentration of exoprotease in step (2) is 1-5% o by weight of the yeast-containing material.
29. The method according to claim 20, wherein the concentration of exoprotease in step (2) is 1-5% o by weight of the yeast-containing material.
30. The method according to any one of claims 1 to 12, wherein the concentration of exoprotease in step (2) is 1 to 2% o by weight of the yeast-containing raw material.
31. A method according to any one of claims 1 to 12, wherein the amount of exoprotease is 0.2 to 1U per 1kg of yeast containing material.
32. The method according to claim 14, wherein the amount of exoprotease is 0.2-1U per 1kg of yeast-containing raw material.
33. The method of claim 16, wherein the amount of exoprotease is 0.2-1U per 1kg of yeast-containing material.
34. The method of claim 17, wherein the amount of exoprotease is 0.2-1U per 1kg of yeast-containing material.
35. The method of claim 20, wherein the amount of exoprotease is 0.2-1U per 1kg of yeast-containing material.
36. The method of any one of claims 1 to 12, wherein the exoprotease is subjected to the enzymatic hydrolysis at a temperature of 60 to 70 ℃, at a pH of 7.0 to 8.0, and for a time of 3 to 6 hours.
37. The method of claim 14, wherein the exoprotease is enzymatically hydrolyzed at 60-70 deg.C, pH7.0-8.0, and time 3-6 h.
38. The method of claim 16, wherein the exoprotease is enzymatically hydrolyzed at 60-70 deg.C, pH7.0-8.0, and time 3-6 h.
39. The method of claim 17, wherein the exoprotease is enzymatically hydrolyzed at 60-70 deg.C, pH7.0-8.0, and time 3-6 h.
40. The method of claim 20, wherein the exoprotease is enzymatically hydrolyzed at 60-70 deg.C, pH7.0-8.0, and time 3-6 h.
41. The method of claim 25, wherein the exoprotease is at a temperature of 60-70 ℃, a pH of 7.0-8.0, and an enzymatic time of 3-6 hours.
42. The method according to any one of claims 1-12, further comprising: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
43. The method of claim 14, further comprising: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
44. The method of claim 16, further comprising: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
45. The method of claim 17, further comprising: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
46. The method of claim 20, further comprising: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
47. The method of claim 25, further comprising: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
48. The method of claim 31, further comprising: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
49. The method of claim 36, further comprising: and (3) centrifuging the solution obtained by enzymolysis in the step (2) to obtain a nucleic acid extracting solution.
50. The method of claim 42, wherein the nucleic acid extract is filtered to obtain the extracted nucleic acid solution.
51. The method of any one of claims 43 to 49, wherein the nucleic acid extract is subjected to membrane filtration to obtain an extracted nucleic acid solution.
52. The method according to any one of claims 1 to 12, wherein the yeast-containing material contains 20% by weight or more of nucleic acid with an RNA of more than 12% by weight based on 100% by weight of baker's yeast cells.
53. The method according to claim 14, wherein the yeast-containing material contains 20% by weight or more of nucleic acid and the RNA content is more than 12% by weight based on 100% by weight of the baker's yeast cell.
54. The method according to claim 16, wherein the yeast-containing material contains 20% by weight or more of nucleic acid and the RNA content is more than 12% by weight based on 100% by weight of the baker's yeast cell.
55. The method according to claim 17, wherein the yeast-containing material contains 20% by weight or more of nucleic acid and the RNA content is more than 12% by weight based on 100% by weight of the baker's yeast cell.
56. The method according to claim 20, wherein the yeast-containing material contains 20% by weight or more of nucleic acid and the RNA content is more than 12% by weight based on 100% by weight of the baker's yeast cell.
57. The method according to claim 25, wherein the yeast-containing material contains 20% by weight or more of nucleic acid and the RNA content is more than 12% by weight based on 100% by weight of the baker's yeast cell.
58. The method according to claim 31, wherein the yeast-containing material contains 20% by weight or more of nucleic acid and the RNA content is more than 12% by weight based on 100% by weight of the baker's yeast cell.
59. The method according to claim 36, wherein the yeast-containing material contains 20% by weight or more of nucleic acid and the RNA content is more than 12% by weight based on 100% by weight of the baker's yeast cell.
60. The method according to claim 49, wherein the yeast-containing material contains 20% by weight or more of nucleic acid and the RNA content is more than 12% by weight based on 100% by weight of the baker's yeast cells.
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