AU2021107435A4

AU2021107435A4 - Method for extracting edible fish sperm DNA

Info

Publication number: AU2021107435A4
Application number: AU2021107435A
Authority: AU
Inventors: Ping Dong; Jingfeng Wang; Yuhan Xue
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-07-15
Filing date: 2021-08-25
Publication date: 2021-12-23
Anticipated expiration: 2029-08-25
Also published as: CN113416728A

Abstract

The present disclosure provides a method for extracting edible fish sperm DNA. The method comprises washing fish sperm as a raw material with a buffer solution; adding the fish sperm as the raw material into the buffer solution, and homogenizing and centrifuging to obtain a precipitate with an approximate skin color; dissolving the precipitate with a concentrated salt solution to form a solution; adjusting pH of the solution to pH 9-11 with an alkaline solution, and centrifuging to obtain a supernatant; adjusting pH of the supernatant to pH 6-7 with an acid solution to obtain a supernatant containing DNA; precipitating with 50~100% ethanol to obtain a fish sperm DNA precipitate; dissolving the fish sperm DNA precipitate with sterile deionized water and purifying. The method for extracting edible fish sperm DNA provided by the present disclosure avoids using toxic and harmful reagents in other methods, and the obtained fish sperm DNA is safe and edible, has high purity, and the method has a high recovery rate, can perform extraction and production on a large scale, and is more suitable for use in animals and clinical research, thereby providing enriched the application range of the fish sperm DNA.

Description

METHOD FOR EXTRACTING EDIBLE FISH SPERM DNA TECHNICAL FIELD

The present disclosure relates to a technical field of combining food, marine organisms and

medicine, and specifically relates to a method for extracting edible fish sperm DNA.

BACKGROUND

In a trend of increasingly serious population aging in China and in context of the limited

medical resources that cannot satisfy increasing medical needs, it is urgent to strengthen

pre-clinical intervention and home care, and precision medicine and precision nutrition are

also on the agenda. Nucleic acids constitute material basis of genetic information and are

important research contents of precision medicine and precision nutrition. According to

reports, dietary nucleic acids have physiological functions mainly comprising promoting

growth and development, regulating liver function, anti-oxidation; relieving physical fatigue,

and assisting in improving memory, which have a wide range of applications in cosmetics,

food, and health products.

Dietary nucleic acids contain a large proportion of RNA in most foods, while DNA is

generally rich in sperm-riched substances, such as fish sperm and pollen. The existing DNA

products are mainly fish sperm DNA and calf thymus DNA. The fish spermary, which is

used as waste during processing, is rich in DNA, and the fish sperm DNA has been proved

to have functions such as skin maintenance and anemia treatment. Thus, methord that

effectively extract DNA from fish spermary is very important.

However, during research on existing methods extracting fish sperm DNA, it is found

that the extraction process mostly involves use of toxic and harmful reagents, thereby

hindering its application in fields of food, health products, cosmetics and so on. WANG Jian

(Extracting Nucleic Acid from Marine Fish Sperm, Journal of Wuxi University of Light

Industry (Food and Biotechnology), Issue 2, 2003, pp. 71-74), TANG Xiaoli (Fast and

pollution-free extraction process of fish sperm DNA, Guangzhou Chemical Industry, 1996

Volume 24, Issue 4), LIU Runzhi (Research on Extraction of Deoxyribonucleic Acid (DNA)

from Carp Spermary, Journal of Natural Science of Hunan Normal University, Vol. 11,

Issue 3, 1988) recite that extraction rate of fish sperm DNA was 6-25%, but sodium dodecyl sulfate (SDS) was used in experiments to denature the protein. Patent CN110760509A uses filter membranes with different molecular weight cut-offs to obtain pure fish sperm DNA and protamine at the same time, but does not analyze purity and recovery rate of the obtained DNA.

To sum up, most of the existing methods use denaturants such as phenol,chloroform or

SDS, which do not correspond to requirements of green processing technology, energy

saving and emission reduction. A new and healthier method for extracting complete strip,

high purity, edible fish sperm DNA is desirable.

SUMMARY

The purpose of the present disclosure is to provide a method for extracting edible fish sperm

DNA to overcome the shortcomings of the prior art.

In order to achieve the above purpose, the present disclosure adopts the specific technical

solutions as follows.

The method for extracting edible fish sperm DNA includes the following steps:

(1) washing a fish sperm as a raw material with a buffer solution ;

(2) adding the fish sperm as the raw material processed in (1) into the buffer solution,

and homogenizing and centrifuging to obtain a precipitate with an approximate skin color;

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

solution;

(4) adjusting pH of the solution in (3) to pH 9-11 with an alkaline solution, and

centrifuging to obtain a supernatant;

(5) adjusting pH of the supernatant in (4) to pH 6-7 with an acid solution to obtain a

supernatant containing DNA;

(6) precipitating with 50~100% ethanol to obtain a fish sperm DNA precipitate;

(7) dissolving the fish sperm DNA precipitate in (6) with sterile deionized water and

purifying.

Further, the fish can be selected as walleye pollack which is relatively easy to obtain in life.

Further, the buffer solution used in the step (1) is an SSC solution, specifically a mixture of

0.05-1 M sodium chloride and 0.01-0.1 M sodium citrate (14:5, c/c).

Further, the specific procedure of the step (2) is: adding a 2 times the weight of the pre-cooled SSC solution compared to the raw material and homogenizing for 1.5 min; centrifuging at 4000 rpm for 15 min at 4C and discarding the supernatant; transfering the precipitate to 2 times the weight of the pre-cooled SSC solution compared to the raw material and homogenizing for 1 min; and centrifuging at 4000 rpm for 15 min at 4C, discarding the supernatant, with two replications. The specific operation of the centrifugation has been verified by many experiments, and it can bring about a better centrifugal effect.

Further, the concentrated salt solution in the step (3) is specifically selected as a 10-20 wt%

sodium chloride solution; the specific operation is adding a 10 times the weight of 10-20

wt% sodium chloride solution compared to the raw material, stirring to fully dissolving a

fish sperm desoxyribose nucleoprotein(DNP), and standing at 4C for 48 h.

Further, in the step (4), the alkaline solution is prepared by sodium carbonate, sodium

bicarbonate or a mixture of both; specifically, by adding the alkaline solution, adjusting pH of the

DNP solution according to amino acid composition within a general adjustment range of pH 9 -11.

Taking extraction of walleye pollack sperm as an example, the pH of the DNP solution is adjusted

to pH 9.7; the precipitate obtained by centrifugation at 10,000 rpm at 4C for 10 min is discarded;

alkaline solution is added to adjust the pH of the DNP solution to pH 10.8; the precipitate

obtained by centrifugation at 10,000 rpm at 4C for 10 min is discarded.

Further, in the step (5), the acidic solution is a mixture of one or more of hydrochloric acid,

acetic acid, and citric acid, the pH of the DNP solution is adjusted to pH 6.5; and the

precipitate obtained by centrifugation at 10,000 rpm at 4C for 10 min is discarded.

The specific operation of the step (6) includes: adding 2 times volume of pre-cooled

-100% ethanol,centrifuging at 10,000 rpm at 4C for 10 min, and discarding the

supernatant, with two replications; washing the precipitate with pre-cooled 50-100% ethanol

twice, and air-drying to remove ethanol.

Further, in the step (7), purification is performed by an ultrafiltration membrane having a

molecular weight cut-off of 5000.

Advantages and beneficial effects of the present disclosure:

The present disclosure uses sodium chloride, edible alkaline solution, hydrochloric acid and

other solutions instead of phenol, chloroform, sodium dodecyl sulfate (SDS) to obtain fish sperm

DNA, providing a safe method for extracting edible fish sperm DNA.

The method for extracting fish sperm DNA provided by the present disclosure avoids using phenol, chloroform, SDS and other reagents in other methods, and the obtained fish sperm DNA is safe and edible, has high purity. The method has a higher recovery rate, and can perform extraction and production on a large scale and is more suitable for animal and clinical research, providing a enriched application range of fish sperm DNA, especially is suitable for the extraction of marine fish-walleye pollack sperm.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic flow chart of the method for extracting edible walleye pollack

sperm DNA in Examples.

Fig. 2 is a graph showing the results of analysis of the amino acid composition and

content of walleye pollack used in Examples.

Fig. 3 is a absorbance-change curve of DNA obtained by the extraction method in

Examples at different wavelengths .

Fig. 4 is a graph showing results of agarose gel electrophoresis for DNA obtained by the

extraction method in Examples.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure will be further explained by way of Examples below, but the

present disclosure is not limited to the scope of the described Examples. In the following

Examples, experimental methods without specific conditions are selected according to

conventional methods and conditions, or according to product specification.

The numerical values disclosed in the Examples of the present disclosure are

approximate values, rather than definite values. Where error or experimental conditions are

allowable, all values within the error range can be included and are not limited to the

specific values disclosed in Examples of the present disclosure.

Example 1:

In this Example, amino acid analysis of the fish sperm is used to determine pH of the

alkaline solution adjustment solution. For the amino acid analysis, referring to the method

described in GB 5009.124-2016 "Determination of Amino Acids in Foods in National Food

Safety Standard", the method includes:

(1) thawing the fish sperm, homogenizing for 1 min, with 3 times repetition;

(2) taking an appropriate amount of the fish sperm, homogenizing to determine the

protein concentration in the walleye pollack sperm, specifically including:

taking 1 mL of the fish sperm to homogenize and adding 3 mL of PBS buffer to dilute,

and recording as "M";

taking 200 L of the homogenate of "M4" and adding 200 L of PBS buffer to dilute, repeating the operation to dilute the homogenate of the walleye pollack sperm to 4 times, 8

times, 16 times, 32 times and 64 times, and recording as "M4 ", "M8", "M16", "M32", "M64", respectively;

adding 20 L of the homogenates of fish sperm having different diluted concentration to

sample wells of a 96-well plate, adding 200 L of BCA working solution, and standing at

37°C for 30 min; and

measuring A562 and calculating the protein concentration of fish sperm, thereby

determining that the mass of the homogenate of fish sperm to be weighed is 0.3501 g.

(3) adding 0.3501 g of the homogenate of the fish sperm to an ampoule, adding 15 mL

of 6 M HCl (containing 5% mercaptoethanol), filling with nitrogen, and sealing the ampoule

by melting with an alcohol lamp;

(4) putting the ampoule into a 110 °C electric heating air-blowing thermostat to

hydrolyze for 22 h, taking out and cooling, and diluting the hydrolysate to 50 mL with 0.02

M HCl;

(5) transferring 1 mL of hydrolysate into an ampoule, placing the ampoule in a water

bath at 60°C, and blowing dry with a compressed air machine.

(6) adding 3-5 drops of ultrapure water, blowing dry and removing the acid, repeating

the operation 3 times; and

(7) redissolving with 2 mL of 0.02 M HCl, filtering through a 0.22 m water-based

filter membrane into a liquid phase bottle as a solution to be tested, and finally analyzing

with Hitachi L-8900 automatic amino acid analyzer.

The results of amino acid analysis of the fish sperm are shown in Figure 2 and Table 1.

The results show that the fish sperm has large proportion of basic amino acids, wherein the

proportions of lysine and arginine proportions of are 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 pH 9-11. According to the property of "the solubility of protein or amino acid at the isoelectric point being the smallest", the pH is finally obtained. Thus, the pH adjustment range when the alkaline solution is used to remove the fish sperm is pH 9-11.

Table 1. Amino acid composition and content analysis for walleye pollack sperm ESTD Pk# RT Name Height Area /1 Conc/ng conc/nmol 1 2.227 Tau 52480 607191 0.418 52.375192 4 4.893 Asp 417182 5227255 3.088 411.039249 5 5.520 Thr 346813 4473658 2.487 296.169766 6 6.140 Ser 331914 4301340 2.348 246.741467 7 6.940 Glu 491867 7676249 4.257 626.214510 9 9.880 Gly 414108 7299470 4.121 309.350475 10 10.840 Ala 286059 7154775 4.115 366.618624 11 12.220 Cys 7382 88257 0.046 11.139554 12 12.900 Val 369330 4778155 2.621 306.900998 13 14.200 Met 47639 950699 0.516 76.989824 15 16.507 Ile 98751 2709809 1.590 208.545397 16 17.513 Leu 250830 5667473 3.541 464.598778 17 18.053 Tyr 117826 1854998 1.099 199.139009 18 18.987 Phe 96014 1883380 1.138 187.974000 21 20.973 Lys 634343 7557830 3.992 583.577225 23 22.413 NH3 379707 7798645 7.585 129.165361 24 23.333 His 88332 1398991 0.792 122.893651 25 27.340 Arg 209344 531575 3.191 555.800731 Totals 4639321 76743902 46.943

Example 2

The Example aims to select appropriate centrifugal conditions.

The centrifugation speed and time are depending on the conditions of the test precipitation. if

the centrifugation speed is too low, the precipitate will float in the solution and cannot be

removed. If the centrifugation speed is too high, it may cause degradation of fish sperm DNA and

affect subsequent experiments. The centrifugation speed involved in the Example is 4000-10,000

rpm, and the time is 10-15 min. The centrifugation conditions for obtaining DNP solution were at

4°C, at 4000 rpm for 15 min; the centrifugation conditions in the step of adjusting the pH of DNP

solution to remove the fish sperm were at 4C at 10,000 rpm for 10 min; the centrifugation

conditions in the ultrafiltration purification step were at 4C at 7000 rpm for 20 min. Under these

conditions, the obtained DNA bands are complete and in high purity.

Example 3

The Example determines the pH range for removing an excess of the alkaline solution.

According to the results shown in Figure 2, the pH of the DNP solution is adjusted to about

3.2 due to the high content of glutamic acid in the protamine, such that the protamine is removed

and at the same time the excess of alkaline solution is neutralized; but the effect of removing the

protein is not significant and under the condition of pH 3.2, DNA may be degraded, thereby the

pH of the DNP solution is adjusted back to the pH before adding the alkaline solution, ranging

from pH 6.0 to pH 7.0 to ensure that the excess of the alkaline solution is neutralized.

Example 4:

In the Example, a spermary tissue of the walleye pollack was used as a raw material, and the

extraction was performed according to the schematic diagram of the method for extracting edible

walleye pollack sperm DNA in FIG. 1. The method includes:

(1) washing the spermary tissue of the walleye pollack by using a SSC solution (a mixed

solution of 0.14 M sodium chloride and 0.05 M sodium citrate), bloting with a filter paper,

weighing, putting into a masher;

(2) adding a 2 times the weight of the pre-cooled SSC solution compared to the raw

material to the walleye pollack spermary obtained in (1), homogenizing for 1.5 min; centrifuging

at 4C at 4000 rpm for 15 min, discarding a supernatant; transferring a precipitate to a 2 times

the weight of the pre-cooled SSC solution compared to the raw material, homogenizing for 1

min; centrifuging at 4000 rpm for 15 min at 4C, discarding a supernatant, and repeating the

operation twice to obtain a precipitate with a approximate skin color;

(3) adding a 10 times the weight of 10% sodium chloride solution compared to the raw

material to the precipitate described in (2), stirring to fully dissolve the precipitate described in

(2), and standing at 4C for 48 hours;

(4) adding a saturated sodium carbonate solution to the solution obtained in (3), adjusting pH

of the DNP solution to pH 9.7, centrifuging at 4C, at 10,000 rpm for 10 min, discarding the

precipitate; adding a saturated sodium carbonate solution, adjusting pH of the DNP solution pH

10.8; centrifuging at 4C, at 10,000 rpm for 10 min, discarding the precipitate, and taking a

supernatant;

(5) adding hydrochloric acid to the supernatant in the step (5), adjusting pH of the DNP

solution to pH 6.5; centrifuging at 10,000 rpm at 4C for 10 min, discarding a precipitate, and

taking a supernatant; wherein the pH involved in steps (4) and (5) is determined according to the amino acid composition and content of the fish sperm of the used walleye pollack; and Figure 2 is a graph of the amino acid composition and content analysis of the walleye pollack used in the present disclosure.

(6) adding the supernatant obtained in (5) to 2 times volume of pre-cooled 75% ethanol,

centrifuging at 10,000 rpm at 4C for 10 min, discarding a supernatant, and repeating the

operation twice; washing the precipitate with absolute ethanol twice, air-drying to remove

ethanol, and dissolving the DNA in sterile deionized water to obtain a DNA solution; and

(7) transferring the solution in (6) to an ultrafiltration tube having a molecular weight cut-off

of 5000, and centrifuging at 7000 rpm for 20 minutes to purify the DNA.

The method for evaluating the content and purity of the DNA extracted in the Examples of

the present disclosure is as follows.

(1) analysis of recovery rate

The recovery rate of DNA extracted in Examples of the present disclosure can reach 1-5%,

and the specific operation and calculation method are as follows.

In the present disclosure, the DNA recovery rate refers to percentage of dry weight of the

extracted fish sperm DNA to dry weight of the fish sperm. The method for calculating the dry

weight of the fish sperm is measured by measuring a moisture content of the fish sperm, referring

to GB 5009.3-2016 "Determination of Moisture in Foods in National Food Safety Standards".

The fish sperm in the present disclosure is suitably determined using a two-step drying method in

a direct drying method. The specific method is as follows:

weighing 2-5 g of clean fish sperm (with the accuracy to 0.0001 g), recording as "ml", and

air-drying for 15-20 h, crushing and mixing the air-dried sample;

taking a flat weighing bottle made of clean glass and placing in a drying oven at 101-105°C

for 1 h, covering, taking out and putting in a desiccator, and cooling for 0.5 h, and weighing;

repeating the drying operation until mass difference between two drying processes as before

and after is < 2 mg, and the last weighed value is recorded as "m5";

weighing the air-dried fish sperm and recording as "m2", putting into a dry weighing bottle

to make sure that the sample thickness is <5 mm, weighing and recording as "m3;

capping and putting in a drying oven at 101-105°C, and placing the cap on the side of the

bottle obliquely, drying for 2-4 hours, after capping, taking out and cooling in a desiccator for 0.5

h, and weighing; capping and putting in a drying oven at 101-105°C, and placing the cap on the side of the bottle obliquely, drying for 1 hour, after capping, taking out and cooling in a desiccator for 0.5 h, and weighing; repeating the drying operation until mass difference between two drying processes as before and after is < 2 mg, and the last weighed value is recorded as "m4";

Calculation formula of the moisture content:

m1 -m2 +m2 xm3 -m4 m3 - m5 X 100% m1 Upon calculating as described above, the recovery rate of the fish sperm DNA in the method

of the present disclosure is 1-5%. In the research on extracting deoxyribonucleic acid (DNA)

from a spermary in the prior art, the DNA extraction rate is about 8-10%, but during the

extraction process, the addition of sodium dodecyl sulfate (SDS) may contaminate the obtained

fish sperm DNA, which is also a technical problem to be solved by the present disclosure; the

recovery rate of the fish sperm DNA in the present Examples and the yields of the fish sperm

DNA-Na and calf thymus DNA described in the patent CN106031709B and Xu Zhipeng et al

(Study on the Process of Large-scale Preparation of Calf Thymus DNA, Pharmaceutical

Biotechnology, 2013 Vol. 20, Issue 5, pp. 435-438) are similar, while the fish sperm DNA

prepared by the latter two methods are non-edible. That is, the present disclosure can be used for

subsequent large-scale preparation and activity studies of the fish sperm DNA.

(2) Purity analysis

2 L of the DNA solution obtained in step (6) is taken and placed in Nano-100 for detection

to obtain an absorbance change curve of the DNA obtained by the method according to the

Examples of the present disclosure at different wavelengths in Fig. 3, and the purity of the

obtained DNA is calculated. The calculation formula is as follows.

(3) Purity = A260/A280

As shown in Figure 3, A260/A280 of the extracted walleye pollack sperm DNA is 1.80,

which can confirm its high purity, proving that this Example can obtain a high-purity DNA with

low contamination of protein.

7.5 pL of the DNA solution obtained in step (6) is taken and mixed evenly with 1.5 L of 6x

loading buffer. 6 L of the sample is taken, and electrophoresis is performed on a 1% agarose gel

for 1 h at a voltage of 120 V. The detection result of the agarose gel electrophoresis is shown in

Fig. 4, the first lane is the fish sperm DNA extracted in this Example and the band is complete,

and the molecular size is between 10kb and 15kb, which can meet requirements of subsequent

activity analysis.

The foregoing descriptions are only preferred examples of the present disclosure, and do not

constitute a limitation to the scope of the claims. Other substantially equivalent alternatives that

can be envisaged by those skilled in the art are all within the protection scope of the present

disclosure.

Claims

What is claimed is:

1. A method for extracting edible fish sperm DNA, comprising the following steps:

(1) washing a fish sperm as a raw material with a buffer solution ;

solution;

centrifuging to obtain a supernatant;

supernatant containing DNA;

(6) precipitating with 50-100% ethanol to obtain a fish sperm DNA precipitate;

purifying.

2. The method for extracting edible fish sperm DNA according to claim 1, wherein the

buffer solution used in the step (1) is an SSC solution, which is specifically a mixed solution

of 0.05-1 M sodium chloride and 0.01-0.1 M sodium citrate with a concentration ratio of

14:5.

3. The method for extracting edible fish sperm DNA according to claim 1, wherein the

specific procedure of the step (2) is: adding a 2 times the weight of the pre-cooled SSC

solution compared to the raw material and homogenizing for 1.5 min; centrifuging at 4000

rpm for 15 min at 4C and discarding the supernatant; transfering the precipitate to 2 times

the weight of the pre-cooled SSC solution compared to the raw material and homogenizing

for 1 min; and centrifuging at 4000 rpm for 15 min at 4C, discarding the supernatant, with

two replications.

4. The method for extracting edible fish sperm DNA according to claim 1, wherein the

concentrated salt solution in the step (3) is specifically selected as a 10-20 wt% sodium

chloride solution; the specific operation is adding a 10 times the weight of 10-20 wt%

sodium chloride solution compared to the raw material, stirring to fully dissolving a fish

sperm DNP(desoxyribose nucleoprotein), and standing at 4C for 48 h.

5. The method for extracting edible fish sperm DNA according to claim 1, wherein the

alkaline solution in the step (4) is prepared by sodium carbonate, sodium bicarbonate or a

mixture of the sodium carbonate and sodium bicarbonate; specifically, by adding the

alkaline solution, adjusting pH of the DNP solution according to amino acid composition

within a general adjustment range of pH 9-11.

6. The method for extracting edible fish sperm DNA according to claim 5, wherein the

pH of the DNP solution is adjusted to pH 9.7; the precipitate obtained by centrifugation at

,000 rpm at 4C for 10 min is discarded; an alkaline solution is added to adjust the pH of

the DNP solution to pH 10.8; and the precipitate obtained by centrifugation at 10,000 rpm at

4°C for 10 min is discarded.

7. The method for extracting edible fish sperm DNA according to claim 1, wherein the

acidic solution in the step (5) is one or more of hydrochloric acid, acetic acid, and citric acid;

the pH of the DNP solution is adjusted to pH 6.5; and the precipitate obtained by

centrifugation at 10,000 rpm at 4C for 10 min is discarded.

8. The method for extracting edible fish sperm DNA according to claim 1, wherein the

specific operation of the step (6) comprises adding 2 times volume of pre-cooled 50-100%

ethanol, centrifuging at 10,000 rpm at 4C for 10 min, and discarding the supernatant, with

two replications; washing the precipitate with pre-cooled 50-100% ethanol twice, and

air-drying to remove ethanol.

9. The method for extracting edible fish sperm DNA according to claim 1, wherein in

the step (7), purification is performed by an ultrafiltration membrane having a molecular

weight cut-off of 5000.

10. The method for extracting edible fish sperm DNA according to claim 1, wherein the

fish is selected as a marine fish, specifically walleye pollack.