阅读说明：本技术 一种可食用鱼精dna的提取方法 (Method for extracting edible milt DNA ) 是由 董平 薛聿函 王静凤 于 2021-07-15 设计创作，主要内容包括：本发明提供了一种可食用鱼精DNA的提取方法,该方法包括：使用缓冲溶液清洗鱼精原料；将经过处理后的鱼精原料投入缓冲溶液中匀浆、离心,得到接近肤色的沉淀；用浓盐溶液溶解获得的沉淀物形成溶液；用碱性溶液调节溶液pH至9～11,然后离心得到上清液；用酸性溶液将上清液pH调至6.0～7.0,获得含DNA的上清液；用浓度为50～100%乙醇沉淀得到鱼精DNA沉淀；用无菌去离子水溶解鱼精DNA沉淀,并进行纯化,即可。本发明提供的鱼精DNA提取方法规避了其他方法中有毒有害试剂的使用,获得的鱼精DNA安全可食用,纯度高,且有较高回收率,能够规模化提取生产,更适合用于动物及临床研究,丰富了鱼精DNA的应用范围。(The invention provides an extraction method of edible milt DNA, which comprises the following steps: washing the fish extract raw material by using a buffer solution; putting the processed milt raw material into a buffer solution for homogenate and centrifugation to obtain a precipitate close to skin color; dissolving the obtained precipitate with concentrated salt solution to form solution; adjusting the pH of the solution to 9-11 by using an alkaline solution, and centrifuging to obtain a supernatant; adjusting the pH of the supernatant to 6.0-7.0 by using an acid solution to obtain a supernatant containing DNA; precipitating with 50-100% ethanol to obtain protamine DNA precipitate; dissolving the protamine DNA precipitate with sterile deionized water, and purifying. The method for extracting the protamine DNA avoids the use of toxic and harmful reagents in other methods, and the obtained protamine DNA is safe and edible, has high purity and higher recovery rate, can be extracted and produced in a large scale, is more suitable for animal and clinical research, and enriches the application range of the protamine DNA.)

1. A method for extracting edible milt DNA, which is characterized by comprising the following steps:

(1) washing the fish extract raw material by using a buffer solution;

(2) putting the milt raw material treated in the step (1) into a buffer solution for homogenate and centrifugation to obtain a precipitate close to skin color;

(3) dissolving the precipitate obtained in the step (2) with a concentrated salt solution to form a solution;

(4) adjusting the pH value of the solution in the step (3) to 9-11 by using an alkaline solution, and then centrifuging to obtain a supernatant;

(5) adjusting the pH of the supernatant in the step (4) to 6-7 by using an acidic solution to obtain a supernatant containing DNA;

(6) precipitating with 50-100% ethanol to obtain protamine DNA precipitate;

(7) and (4) dissolving the protamine DNA precipitate in the step (6) by using sterile deionized water, and purifying to obtain the fish sperm DNA precipitate.

2. The method for extracting edible milt DNA according to claim 1, wherein the buffer solution used in step (1) is SSC solution, specifically a mixture of 0.05-1M sodium chloride and 0.01-0.1M sodium citrate at a concentration ratio of 14: 5.

3. The method for extracting edible milt DNA according to claim 1, wherein the step (2) comprises the following steps: adding pre-cooled SSC solution 2 times the weight of the raw materials, and homogenizing for 1.5 min; centrifuging at 4 deg.C and 4000 rpm for 15 min, and removing supernatant; transferring the precipitate to SSC solution with 2 times of the weight of the raw materials after precooling, and homogenizing for 1 min; centrifuging at 4000 rpm for 15 min at 4 deg.C, discarding the supernatant, and repeating the operation 2 times.

4. The method for extracting edible milt DNA according to claim 1, wherein the concentrated salt solution in the step (3) is selected from 10-20wt% sodium chloride solution; the specific operation is to add 10-20wt% sodium chloride solution of 10 times of the weight of the raw materials, stir to fully dissolve the protamine deoxyribonuclein, and stand for 48 h at 4 ℃.

5. The method for extracting edible milt DNA according to claim 1, wherein in the step (4), the alkaline solution is prepared from sodium carbonate, sodium bicarbonate or a mixture of the sodium carbonate and the sodium bicarbonate; the method specifically comprises the following steps: the DNP solution is adjusted to the amino acid composition by adding an alkaline solution, typically to a pH of 9-11.

6. The method for extracting edible milt DNA according to claim 5, wherein the pH of DNP solution is adjusted to 9.7; centrifuging at 4 deg.C and 10000 rpm for 10 min, and removing precipitate; adding an alkali solution, and adjusting the pH value of the DNP solution to 10.8; centrifuging at 4 deg.C and 10000 rpm for 10 min, and removing precipitate.

7. The method for extracting edible protamine DNA as claimed in claim 1, wherein the acidic solution in step (5) is one or more of hydrochloric acid, acetic acid and citric acid, and the pH of DNP solution is adjusted to 6.5; centrifuging at 4 deg.C and 10000 rpm for 10 min, and removing precipitate.

8. The method for extracting edible milt DNA according to claim 1, wherein the specific operation of step (6) comprises: adding 2 times volume of precooled 50-100% ethanol, centrifuging at 4 deg.C and 10000 rpm for 10 min, discarding supernatant, and repeating the above operation for 2 times; the pellet was washed 2 times with pre-cooled 50-100% ethanol air dried.

9. The method for extracting edible milt DNA according to claim 1, wherein the step (7) is carried out by purifying through ultrafiltration membrane with molecular weight cut-off of 5000.

10. The method for extracting edible milt DNA according to claim 1, wherein the fish is selected from marine fish, specifically walleye pollack.

Technical Field

The invention relates to the technical field of food, marine organism and medicine combination, in particular to a method for extracting edible milt DNA.

Background

Under the background that the aging trend of the population of China is increasingly serious and limited medical resources are difficult to meet the increasing medical requirements, the pre-disease intervention and the family care are urgently strengthened, and the schedule of accurate medical treatment and accurate nutrition is also increased. Nucleic acid substances are the material basis for constituting genetic information and are important research contents of precise medical treatment and precise nutrition. The physiological functions of dietary nucleic acids are reported to be mainly: promoting growth and development; regulating liver function; oxidation resistance; physical fatigue is relieved; the product can assist in improving memory, and can be widely applied to cosmetics, foods, health products and the like.

Dietary nucleic acids contain RNA in large proportions in most foods, while DNA is generally abundant in sperm-rich materials, such as fish sperm and pollen. The existing DNA products mainly comprise fish sperm DNA and calf thymus DNA, the fish spermary which is taken as waste in the processing process contains abundant DNA, and the fish sperm DNA is proved to have the functions of skin care, anemia treatment and the like, so the method for effectively extracting the DNA from the fish spermary is very important.

However, the research on the existing extraction method of the milt DNA shows that the extraction process mostly involves the use of toxic and harmful reagents, which hinders the application of the milt DNA in the fields of food, health care products, cosmetics and the like. The extraction rate of the DNA of the milt is 6-25% in Wangjian (nucleic acid is extracted from the fish white of a sea fish, Wuxi Ministry university (food and biotechnology), No. 2-74 pages in 2003), Tangxiaoling (rapid and pollution-free extraction process of the DNA of the milt, Guangzhou chemical industry, 24 th volume and 4 th volume in 1996), Liu Runzhi (research on extracting deoxyribonucleic acid (DNA) from the spermary of a carp, Nature science university in Hunan, 11 th volume and 3 rd volume in 1988) and the like, but the protein is denatured by Sodium Dodecyl Sulfate (SDS) in the experiment. Patent CN110760509A utilizes filters with different molecular weight cut-off to obtain puffer fish sperm DNA and protamine at the same time, but it does not analyze the purity and recovery rate of the obtained DNA.

In conclusion, most of the existing methods use phenol-imitated or SDS (sodium dodecyl sulfate) denaturants, so that the methods do not meet the trends of green processing, energy conservation and emission reduction, how to avoid the use of toxic and harmful reagents to the greatest extent and obtain edible milt DNA with complete strips and high purity also need a new healthier extraction method.

Disclosure of Invention

The invention aims to provide an extraction method of edible milt DNA, which makes up for the defects of the prior art.

In order to achieve the purpose, the invention adopts the following specific technical scheme:

a method for extracting edible milt DNA, which comprises the following steps:

(1) washing the fish extract raw material by using a buffer solution;

(2) putting the milt raw material treated in the step (1) into a buffer solution for homogenate and centrifugation to obtain a precipitate close to skin color;

(3) dissolving the precipitate obtained in the step (2) with a concentrated salt solution to form a solution;

(4) adjusting the pH value of the solution in the step (3) to 9-11 by using an alkaline solution, and then centrifuging to obtain a supernatant;

(5) adjusting the pH of the supernatant in the step (4) to 6-7 by using an acidic solution to obtain a supernatant containing DNA;

(6) precipitating with 50-100% ethanol to obtain protamine DNA precipitate;

(7) and (4) dissolving the protamine DNA precipitate in the step (6) by using sterile deionized water, and purifying to obtain the fish sperm DNA precipitate.

Further, the fish can be selected from Alaska Pollack, which is a marine fish easily obtained in life.

Further, the buffer solution used in the step (1) is an SSC solution, specifically a mixture of 0.05-1M sodium chloride and 0.01-0.1M sodium citrate (14: 5, c/c).

Further, the specific process of the step (2) is as follows: adding pre-cooled SSC solution 2 times the weight of the raw materials, and homogenizing for 1.5 min; centrifuging at 4 deg.C and 4000 rpm for 15 min, and removing supernatant; transferring the precipitate to SSC solution with 2 times of the weight of the raw materials after precooling, and homogenizing for 1 min; centrifuging at 4 deg.C and 4000 rpm for 15 min, removing supernatant, and repeating the operation for 2 times; the specific operation of the centrifugation is verified by a plurality of experiments, and a better centrifugation effect can be obtained.

Further, the concentrated salt solution in the step (3) is specifically selected to be a 10-20wt% sodium chloride solution; the specific operation is that 10-20wt% sodium chloride solution which is 10 times of the weight of the raw materials is added and stirred to fully dissolve the protamine Deoxyribonuclein (DNP), and the mixture is kept stand for 48 hours at 4 ℃.

Further, the alkaline solution in the step (4) is prepared by sodium carbonate, sodium bicarbonate or a mixture of the sodium carbonate and the sodium bicarbonate; the method specifically comprises the following steps: adding an alkaline solution, and adjusting the DNP solution according to the composition of amino acid, wherein the general adjustment range is pH 9-11; taking the extraction of the walleye pollack milt as an example, adjusting the pH =9.7 of the DNP solution; centrifuging at 4 deg.C and 10000 rpm for 10 min, and removing precipitate; adding an alkali solution, and adjusting the pH =10.8 of the DNP solution; centrifuging at 4 deg.C and 10000 rpm for 10 min, and removing precipitate.

Further, in the step (5), the acidic solution is one or a mixture of hydrochloric acid, acetic acid and citric acid, and the pH =6.5 of the DNP solution is adjusted; centrifuging at 4 deg.C and 10000 rpm for 10 min, and removing precipitate.

The step (6) comprises the following specific operations: adding 2 times volume of precooled 50-100% ethanol, centrifuging at 4 deg.C and 10000 rpm for 10 min, discarding supernatant, and repeating the above operation for 2 times; the pellet was washed 2 times with pre-cooled 50-100% ethanol air dried.

Further, in the step (7), the purification is performed by an ultrafiltration membrane with a molecular weight cut-off of 5000.

The invention has the advantages and beneficial effects that:

the invention uses sodium chloride, edible alkaline solution, hydrochloric acid and other solutions to replace phenol, chloroform, Sodium Dodecyl Sulfate (SDS) and the like to obtain the fish sperm DNA, and provides a safe extraction method of the edible fish sperm DNA.

The method for extracting the milt DNA avoids the use of reagents such as phenol, chloroform, SDS and the like in other methods, the obtained milt DNA is safe and edible, has high purity and higher recovery rate, can be extracted and produced in a large scale, is more suitable for animal and clinical research, enriches the application range of the milt DNA, and is particularly suitable for extracting the milt of the marine fish and the walleye pollack.

Drawings

FIG. 1 is a schematic flow chart of the method for extracting DNA from edible Theragra Chalcogramma extract in the example.

FIG. 2 is a graph showing the results of amino acid composition and content analysis of Alaska pollack sperm used in the examples.

FIG. 3 is a graph showing the change of absorbance at different wavelengths of DNA obtained by the extraction method in the examples.

FIG. 4 is a diagram showing the results of DNA agarose gel electrophoresis obtained by the extraction method in the examples.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The numerical values set forth in the examples of the present invention are approximations, not necessarily values. All values within the error range may be included without limiting to the specific values disclosed in the embodiments of the present invention, where the error or experimental conditions allow.

Example 1:

in this example, amino acid analysis of milt to determine pH of the aqueous alkali solution was carried out according to the method described in GB 5009.124-2016 determination of amino acids in national food Standard for food safety, which comprises:

(1) thawing the fish sperm, homogenizing for 1 min, and repeating the operation for 3 times.

(2) Taking a proper amount of milt homogenate to measure the protein concentration in the walleye pollack milt, and the specific method comprises the following steps:

1 mL of milt homogenate was diluted with 3 mL of PBS buffer and labeled "M4".

200 μ L of homogenate from "M4" was diluted with 200 μ L of PBS buffer, and the procedure was repeated to dilute the Theragra chalcogramma fine homogenate by 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold, labeled "M4", "M8", "M16", "M32", and "M64", respectively.

mu.L of milt homogenate of different dilution times was added to the sample wells of a 96-well plate, and 200. mu.L of BCA working solution was added thereto and left at 37 ℃ for 30 min.

A562 was measured, the protamine protein concentration was calculated and the desired protamine homogenate weight was determined to be 0.3501 g.

(3) An ampoule was filled with 0.3501 g of the fish essence homogenate, 15 mL of 6M HCl (containing 5% mercaptoethanol) was added, nitrogen was added, and the tube was sealed by melting with an alcohol lamp.

(4) And (3) putting the ampoule bottle into an electrothermal blowing constant temperature box at 110 ℃ for hydrolysis for 22 h, taking out and cooling, and metering the volume of the hydrolysate to 50 mL by using 0.02M HCl.

(5) And (3) absorbing 1 mL of hydrolysate in an ampoule bottle, placing in a water bath at 60 ℃, and drying by using a compressed air machine.

(6) Adding 3-5 drops of ultrapure water, drying and discharging acid, and repeating the operation for 3 times.

(7) Redissolving with 2 mL of 0.02M HCl, filtering with 0.22 μ M water system filter membrane to obtain liquid as the solution to be detected, and analyzing with Hitachi L-8900 full-automatic amino acid analyzer.

The results of the protamine amino acid analysis are shown in fig. 2 and table 1, and show that the protamine has a large proportion of basic amino acids, wherein the proportion of lysine and arginine is 11.32% and 10.76%, respectively, the isoelectric point of arginine is pH 10.8, the isoelectric point of lysine is pH 9.7, and the approximate range is pH9-11, and finally the pH is obtained from this, and the pH adjustment range when protamine is removed with an alkali solution is pH 9-pH 11.

TABLE 1 analysis of the amino acid composition and content of Taiyu sperm

Example 2

This example is intended to select suitable centrifugation conditions.

The centrifugal speed and the centrifugal time are determined according to the condition of the sediment to be tested, if the centrifugal speed is too low, the sediment can float in the solution and can not be removed, and if the centrifugal speed is too high, the degradation of the DNA of the milt can be caused, and the subsequent experiment is influenced. The centrifugation speed involved in this example was 4000-10000 rpm for 10-15 min. Centrifuging the DNP solution at 4 ℃ and 4000 rpm for 15 min; centrifuging at 10000 rpm for 10 min at 4 deg.C in the step of adjusting pH of DNP solution to remove protamine; the centrifugation conditions of the ultrafiltration purification step are 4 ℃ and 7000 rpm for 20 min, and under the conditions, the obtained DNA band is complete and has high purity.

Example 3

This example determines the pH range for removing excess alkaline solution.

According to the results shown in FIG. 2, the pH of the DNP solution was adjusted to about 3.2 because of the high content of glutamic acid in protamine, and protamine was removed while neutralizing the excess alkali solution, but the protein removal effect was not significant, and the DNA was likely to be degraded under the condition of pH 3.2, so the pH of the DNP solution was adjusted back to the pH before the addition of the alkali solution, ranging from pH 6.0 to pH 7.0, to ensure that the excess alkali solution was neutralized.

Example 4:

in this example, the extraction is performed according to the schematic diagram of the method for extracting edible walleye pollack sperm DNA in fig. 1, using walleye pollack spermary tissue as raw material, the method includes:

(1) washing sperm tissue of Theragra Chalcogramma with SSC solution (mixed solution of 0.14M sodium chloride and 0.05M sodium citrate), drying with filter paper, and weighing; putting into a mashing machine.

(2) Adding an SSC solution precooled by 2 times of the weight of the raw material into the spermary tissue of the walleye pollack obtained in the step (1), and homogenizing for 1.5 min; centrifuging at 4 deg.C and 4000 rpm for 15 min, and removing supernatant; transferring the precipitate to SSC solution with 2 times of the weight of the raw materials after precooling, and homogenizing for 1 min; centrifuging at 4 deg.C and 4000 rpm for 15 min, discarding supernatant, and repeating the operation for 2 times to obtain precipitate with color similar to skin color.

(3) Adding 10 times of 10% sodium chloride solution of the weight of the raw materials into the precipitate in the step (2), stirring to fully dissolve the precipitate in the step (2), and standing for 48 h at 4 ℃.

(4) Adding a saturated sodium carbonate solution to the solution obtained in (3), adjusting the pH of the DNP solution to =9.7, centrifuging at 4 ℃ and 10000 rpm for 10 min, and discarding the precipitate; adding saturated sodium carbonate solution, and adjusting the pH of the DNP solution to be = 10.8; centrifuging at 4 deg.C and 10000 rpm for 10 min, removing precipitate, and collecting supernatant.

(5) Adding hydrochloric acid into the supernatant obtained in the step (5), and adjusting the pH =6.5 of the DNP solution; centrifuging at 4 deg.C and 10000 rpm for 10 min, removing precipitate, and collecting supernatant. The pH involved in steps (4) and (5) is determined according to the composition and content of the white amino acid of walleye pollack, and FIG. 2 is a graph of the amino acid composition and content analysis of walleye pollack essence adopted in the present invention.

(6) Adding the supernatant obtained in the step (5) into 2 times volume of precooled 75% ethanol, centrifuging at 4 ℃ and 10000 rpm for 10 min, removing the supernatant, and repeating the operation for 2 times; washing the precipitate with pre-cooled absolute ethanol for 2 times, air drying the ethanol, and dissolving the DNA with sterile deionized water to obtain a DNA solution.

(7) The solution in (6) was transferred to an ultrafiltration tube having a molecular weight cut-off of 5000, and centrifuged at 7000 rpm for 20 min to purify the DNA.

The method for evaluating the content and purity of the extracted DNA in the embodiment of the invention comprises the following steps:

(1) recovery analysis

The recovery rate of the DNA extracted by the embodiment of the invention can reach 1-5%, and the specific operation and calculation method comprises the following steps:

the DNA recovery rate in the present invention means the percentage of the dry weight of the DNA extracted from the milt to the dry weight of the milt. The method for calculating the dry weight of the fish extract is measured by measuring the water content of the fish extract, and the water content of the fish extract is measured by a two-step drying method in a direct drying method according to GB 5009.3-2016 (national food safety standard for measuring water in food) < determination of water content in food >:

weighing 2-5 g of clean milt (accurate to 0.0001 g) recorded as m1, naturally drying for 15-20 h, crushing the dried sample, and mixing uniformly.

Taking a flat weighing bottle made of clean glass, placing the flat weighing bottle in a drying box with the temperature of 101-.

The drying operation is repeated until the mass difference between the two times is less than or equal to 2 mg, and the last weighing value is recorded as m 5.

Weighing the dried fish milt as m2, putting the fish milt into a dried weighing bottle to ensure that the thickness of the sample is less than or equal to 5 mm, and weighing as m 3.

Covering and placing in a drying box with temperature of 101-.

Covering and placing in a drying box with the temperature of 101-.

The drying operation is repeated until the mass difference between the two times is less than or equal to 2 mg, and the last weighing value is recorded as m 4.

The calculation formula of the moisture content is as follows:

through the calculation, the recovery rate of the DNA of the milt is 1-5%, the DNA extraction rate is about 8-10% by the research of extracting deoxyribonucleic acid (DNA) from spermary in the prior art, but the possibility of pollution to the obtained milt DNA is caused by adding Sodium Dodecyl Sulfate (SDS) in the extraction process, and the technical problem to be solved by the application is solved; the recovery rate of the sperm DNA in the embodiment is close to the recovery rates of the sperm DNA-Na and the calf thymus DNA described in patents CN106031709B and Xushipeng and the like (technical research on the large-scale preparation of the calf thymus DNA, pharmaceutical biotechnology, 20 vol.20, page 435-438), and the sperm prepared by the two methods is non-edible, namely, the invention can be used for the subsequent large-scale preparation and the activity research of the sperm DNA.

(2) Purity analysis

And (3) placing 2 mu L of the DNA solution obtained in the step (6) in a Nano-100 for detection to obtain a change curve of absorbance of the DNA obtained by the extraction method according to the embodiment of the invention in the figure 3 under different wavelengths, and calculating the purity of the obtained DNA by using the following calculation formula:

purity = A260/A280

As shown in FIG. 3, the purified product of walleye pollack sperm DNAA260/A280 obtained by extraction was 1.80, and it was confirmed that the purity was high, and it was confirmed that this example was able to obtain a high purity DNA with a low degree of protein contamination.

And (3) uniformly mixing 7.5 mu L of the DNA solution obtained in the step (6) with 1.5 mu L of 6 multiplied loading buffer solution, taking 6 mu L of loading, performing electrophoresis for 1 h by using 1% agarose gel at the voltage of 120V, wherein the agarose gel electrophoresis detection result is shown in figure 4, a 1-point sample hole is the protamine DNA extracted in the embodiment, the band is complete, the molecular size is between 10kb and 15kb, and the requirement of subsequent activity analysis can be met.

The above description is only a preferred embodiment of the present invention, and should not be construed as limiting the scope of the claims, and other substantially equivalent alternatives that may be conceived by those skilled in the art are within the scope of the present invention.