CN111066943A - Euphausia superba protein and extraction method thereof - Google Patents
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Abstract
The invention provides euphausia superba protein and an extraction method thereof, wherein the extraction method comprises the following steps: preparing alkaline electrolyzed water: electrolyzing a sodium chloride aqueous solution to obtain alkaline electrolyzed water; mixing Antarctic krill with alkaline electrolyzed water, and homogenizing to obtain a mixture; adjusting the pH value of the mixture to 11.6-12.8, performing ultrasonic treatment, stirring, centrifuging, and taking a supernatant; adjusting the pH of the supernatant to 4-5, centrifuging, and collecting protein precipitate; washing the protein precipitate obtained in the step with double distilled water, adjusting the pH value to 7, and dialyzing to obtain a protein water solution; freeze drying the protein water solution, and degreasing; the mixed solution was centrifuged, and the resulting protein precipitate was dried. The method for extracting the euphausia superba protein by using the alkaline electrolyzed water and ultrasonic assistance has the advantages of simple process, resource saving, environment protection, better solubility, emulsibility, foamability, oil retention property and the like of the obtained protein, and sterilization and anti-oxidation effects on the protein.
Description
Technical Field
The invention relates to an extraction method of euphausia superba protein, and particularly relates to euphausia superba protein and an extraction method thereof.
Background
Antarctic krill is one of invertebrates, belongs to the order of Krill of Crustacea, has a body length of 3-5 cm, is abundant in storage amount, and is conservative and estimated at present to be 6-10 hundred million tons. Protein resources are rich in antarctic bodies, the protein content is 17.56% (the water content of antarctic krill is 77.26%), and the proportion of high-quality protein is high. Amino acid composition analysis of Antarctic krill protein shows that: the antarctic krill protein is rich in amino acid composition, contains all essential amino acids for human bodies, particularly contains lysine which is representative of human nutritional characteristics, and has higher content than that of the well-known tuna, chicken and beef. The antarctic krill protein contains 53% of essential and semi-essential amino acids, and has high nutritive value. Antarctic krill is used as a huge protein library of high-quality marine protein, and has important significance for extracting and developing the protein of the Antarctic krill to supplement animal protein for human in the future.
Electrolyzed water is generally classified into two types, i.e., acidic electrolyzed water and alkaline electrolyzed water. The acidic electrolyzed water generates acid from the anode of the electrode, and is widely used in the fields of medical instrument disinfection, food sterilization, catering service, agricultural fruit and vegetable processing, aquatic product preservation and the like at present; alkaline electrolyzed water can be produced by the negative electrode, and the alkaline electrolyzed water still has higher utilization value as a byproduct for producing acidic electrolyzed water: (1) the alkaline electrolyzed water has the characteristics of easy permeation through cell gaps, strong permeability and the like, so the alkaline electrolyzed water still has strong sterilization capability; (2) the alkaline electrolyzed water has strong reducing power, and is of great help in the aspects of clearing free radicals in vivo and assisting the treatment of various senile diseases (tumors, cardiovascular diseases, diabetes and the like).
Most studies have shown improved quality and safety of Food products after sonication (chandra, Oliver, kentih and ashokumar, 2012) (chandra, j., Oliver, c., kentih, s., & ashokumar, M. (2012). Ultrasonics in Food quality assessment and Food safety. trends in Food Science & Technology,26(2), 88-98.). However, in the prior art, more sodium hydroxide is usually adopted for extracting protein, which is not beneficial to environmental protection; and direct ultrasonic extraction often oxidizes proteins and destroys protein structures. And the protein extracted by the traditional alkali-soluble acid precipitation method has serious protein pollution and poor physical property, is not beneficial to the later processing of food, and has low extraction rate of the protein. Therefore, it is necessary to provide a novel method for extracting protein, which improves the extraction rate of protein and the performance of the extracted protein, and is environmentally friendly.
Disclosure of Invention
The invention aims to overcome the defects of serious pollution, high sodium hydroxide consumption, low extraction rate and the like of the protein extracted by the existing alkali-dissolution acid-precipitation method, and provides a method for extracting the euphausia superba protein by using alkaline electrolyzed water in combination with ultrasound, so that the method not only saves the use amount of sodium hydroxide and improves the protein extraction rate, but also has more excellent properties of the extracted protein.
The invention is realized by the following technical scheme:
an extraction method of euphausia superba protein comprises the following steps:
(1) mixing Antarctic krill with alkaline electrolyzed water, and homogenizing to obtain a mixture; the pH value of the alkaline electrolyzed water is 11.6-12.8, and the alkaline electrolyzed water is obtained by electrolysis of a sodium chloride aqueous solution;
(2) adjusting the pH value of the mixture obtained in the step (1) to 11.6-12.8, performing ultrasonic treatment, stirring, centrifuging and taking a supernatant;
(3) adjusting the pH of the supernatant obtained in the step (2) to 4-5, centrifuging, and collecting protein precipitate;
(4) washing the protein precipitate obtained in the step (3) with double distilled water, adjusting the pH to 7, and dialyzing to obtain a protein water solution;
(5) freeze-drying the protein aqueous solution obtained in the step (4) to obtain antarctic krill protein powder, and then mixing the protein powder with n-hexane for degreasing;
(6) and (5) centrifuging the mixed solution obtained in the step (5), and drying the obtained protein precipitate to obtain the euphausia superba protein.
The concentration of the sodium chloride aqueous solution in the step (1) is 0.5-1.5 wt%, the electrolysis time is 8-12 minutes, and the pH value of the alkaline electrolysis water is preferably 12.0-12.5.
The antarctic krill and alkaline electrolyzed water in the step (1) are mixed according to the feed-liquid ratio of 1g: (5-10) mixing, wherein the homogenization condition is that the mixture is homogenized for 2-5min at the rotating speed of 500-800 r/min.
The ultrasonic power in the step (2) is 300-350W, the ultrasonic time is 20-30min, and the stirring condition is room temperature stirring for 1-2 hours.
The pH value is adjusted by adopting 0.8-1.2mol/L sodium hydroxide aqueous solution in the step (2), and the pH value is adjusted by adopting 2.8-3.2mol/L hydrochloric acid in the step (3).
The centrifugation conditions of the step (2) and the step (3) are 8000-.
The dialysis condition in the step (4) is that the membrane is placed in a 5000-12000Da dialysis bag and is dialyzed for 10 to 24 hours by deionized water.
And (5) mixing the protein powder and n-hexane according to a feed-liquid ratio of 1g: (10-20) mL.
The centrifugation condition in the step (6) is 5000-.
The invention also provides euphausia superba protein extracted by any one of the methods.
The invention has the following advantages and beneficial effects:
(1) the raw materials adopted in the method for extracting the euphausia superba protein by combining the alkaline electrolyzed water and the ultrasonic waves are natural, the protein content is high, and especially the essential amino acid is rich;
(2) the alkaline electrolyzed water used in the method for extracting the euphausia superba protein by combining the alkaline electrolyzed water and the ultrasonic wave has the effective chlorine of 0.35 +/-0.04 mg/L, and also has the characteristics of easiness in penetrating through intercellular gaps, strong permeability and the like, so the alkaline electrolyzed water still has strong sterilization capability, the protein extraction process is also a local killing process, and the protein extracted by the method is safer;
(3) the alkaline electrolyzed water used in the method for extracting the euphausia superba protein by combining the alkaline electrolyzed water and the ultrasonic waves has the oxidation-reduction potential of-840 +/-25.6, has strong reducing power, can prevent the oxidation of the protein in the protein extraction process, and plays a role in protecting the protein;
(4) the alkaline electrolyzed water used in the method for extracting the euphausia superba protein by combining the alkaline electrolyzed water with the ultrasonic waves can reduce the usage amount of sodium hydroxide (the reduction amount is about 33.4%) in the process of extracting the protein, and has better protection effect on the environment;
(5) compared with the protein extracted by the traditional alkali-dissolving acid-precipitation method, the method for extracting the euphausia superba protein by combining the alkaline electrolyzed water and the ultrasonic wave has better physical and chemical properties, is more favorable for processing of later-stage food, improves the high added value of the euphausia superba, and has huge application to future food manufacture;
(6) compared with the traditional alkali-soluble acid precipitation method, the method for extracting the euphausia superba protein by combining the alkaline electrolyzed water and the ultrasonic wave has the advantages of low cost, high protein extraction rate and the like.
Drawings
Figure 1 alkaline electrolyzed water from different concentrations of sodium chloride mixed to give a mixture that requires a volume of 1mol/L sodium hydroxide to adjust ph to 12;
FIG. 2 shows the extraction rate of the protein of the Antarctic krill after the Antarctic krill is treated by ultrasonic with different powers (250- & gt, 350W);
FIG. 3 is a graph showing the particle size of protein extracted from Euphausia superba by ultrasonic time-assisted treatment;
FIG. 4 foaming capacity of ultrasonically assisted extracted proteins;
FIG. 5 scanning electron micrographs of proteins extracted by different ultrasounds (A without ultrasound; B ultrasound for 10 min; C ultrasound for 20 min; D ultrasound for 30 min);
FIG. 6 carbonyl content in proteins extracted with alkaline electrolyzed water and ionic water.
Detailed Description
The invention is further illustrated by the following examples and figures.
Example 1
(1) Preparing alkaline electrolyzed water: preparing sodium chloride aqueous solutions (0.5 wt%, 1 wt%, 1.5 wt%) with different concentrations, and then placing the aqueous solutions in an electrolytic water device (Tianye corporation, Shenkangcuan county, Japan, model FW-200) for electrolysis for 10 minutes to obtain three alkaline electrolytic waters, wherein ORP and pH contents in the three alkaline electrolytic waters are shown in Table 1;
(2) and (3) mixing the unfrozen antarctic krill with alkaline electrolytic water according to the feed-liquid ratio of 1g: 5mL of the mixture is mixed, and then the mixture is homogenized for 2min at the rotating speed of 500r/min to obtain a mixture;
(3) adjusting the pH value of the mixture obtained in the step (2) to 12 by using 1mol/L sodium hydroxide, performing ultrasonic treatment for 20min under the power of 300W, stirring for 2 hours at room temperature, and centrifuging for 10min at 8000r/min to obtain a supernatant;
(4) regulating the pH value of the supernatant obtained in the step (3) to 4.5 by using 3mol/L hydrochloric acid, and centrifuging at the rotating speed of 8000r/min for 10min to collect protein precipitate;
(5) washing the protein precipitate obtained in the step (4) with double distilled water, adjusting the pH to 7 with 1mol/L NaOH, and then putting the protein precipitate into a dialysis bag of 5000Da for dialysis for 12 hours with deionized water to obtain a protein water solution;
(6) freeze-drying the protein aqueous solution obtained in the step (5) to obtain antarctic krill protein powder, and then mixing the protein powder and n-hexane according to a feed-liquid ratio of 1g:15mL for degreasing;
(7) and (4) centrifuging the mixed solution obtained in the step (6) at 6000r/min for 10min, and drying the obtained protein precipitate to obtain the euphausia superba protein.
In the embodiment, the electrolyzed water with different sodium chloride concentrations is used for extracting the proteins of the antarctic krill, and the electrolyzed water with different sodium chloride concentrations is used for comparing and verifying the extracted proteins. The results are shown in table 1: there was no statistical difference in effective reduction potential and pH for alkaline electrolyzed water prepared at different sodium chloride concentrations. The pH intensity of the electrolyzed water is weak, the pH value of the aqueous solution of the mixture is reduced to about 9.5 after the alkaline electrolyzed water and the antarctic krill are homogenized together in the process of extracting the antarctic krill protein, and in order to achieve the effect of extracting the protein by the alkaline-solution acid-precipitation method, the pH value of the homogenized mixture of the antarctic krill needs to be adjusted to 12 by 1mol/L of sodium hydroxide. The use amount of sodium hydroxide can be greatly reduced by adopting alkaline electrolyzed water to replace deionized water to be mixed with shrimp meat, and the volume of 1mol/L of sodium hydroxide is required for adjusting the pH value of a mixture obtained by mixing Antarctic krill with 1L of deionized water and 1L of alkaline electrolyzed water obtained by sodium chloride with different concentrations to 12, as shown in figure 1, the use amount of the sodium hydroxide can be reduced by about 33.4%.
TABLE 1 ORP (Oxidation reduction potential) and pH content in alkaline electrolyzed water obtained with different sodium chloride concentrations
Example 2
(1) Preparing alkaline electrolyzed water: preparing 0.5 wt% sodium chloride water solution, and electrolyzing in water electrolysis device (model FW-200 of Tianye, Shenchuan county, Japan) for 10min to obtain alkaline (pH 12.31 + -0.21) electrolyzed water;
(2) and (3) mixing the unfrozen antarctic krill with alkaline electrolytic water according to the feed-liquid ratio of 1g: mixing 10mL of the mixture, and homogenizing the mixture for 3min at the rotating speed of 800r/min to obtain a mixture;
(3) adjusting the pH value of the mixture obtained in the step (2) to 12 by using 1mol/L sodium hydroxide, placing the mixture under different ultrasonic intensities (250, 275, 300, 325 and 350W of power for ultrasonic treatment for 15min, stirring the mixture at room temperature for 1.5 h, and centrifuging the mixture at 10000r/min for 12min to obtain a supernatant;
(4) regulating the pH of the supernatant obtained in the step (3) to 4.5 by using 3mol/L hydrochloric acid, and centrifuging for 15min at the rotating speed of 10000r/min to collect protein precipitate;
(5) washing the protein precipitate obtained in the step (4) with double distilled water, adjusting the pH to 7 with 1mol/L NaOH, and then putting the protein precipitate into a dialysis bag of 5000Da for dialysis for 12 hours with deionized water to obtain a protein water solution;
(6) freeze-drying the protein aqueous solution obtained in the step (5) to obtain antarctic krill protein powder, and then mixing the protein powder and n-hexane according to a feed-liquid ratio of 1g:10mL for degreasing;
(7) and (4) centrifuging the mixed solution obtained in the step (6) at 6000r/min for 10min, and drying the obtained protein precipitate to obtain the euphausia superba protein.
In the embodiment, ultrasonic treatment with the intensity (250-350W) is adopted to assist in extracting the proteins of the Antarctic krill, and the influence of ultrasonic treatment with different powers on the extraction rate of the extracted proteins is influenced. The results are shown in FIG. 2: effect of different power ultrasonication on extraction rate of protein from Euphausia superba. Generally speaking, the ultrasonic treatment increases the extraction rate of the protein, but the optimal power is 300- & ltSUB & gt 350W.
Example 3
(1) Preparing alkaline electrolyzed water: preparing 1 wt% sodium chloride aqueous solution, and electrolyzing in an electrolytic water device (model FW-200, Tianye, Shenchuan county, Japan) for 10min to obtain alkaline electrolytic water (pH 12.29 + -0.38);
(2) and (3) mixing the unfrozen antarctic krill with alkaline electrolytic water according to the feed-liquid ratio of 1g: mixing 10mL of the mixture, and homogenizing the mixture for 3min at the rotating speed of 800r/min to obtain a mixture;
(3) adjusting the pH value of the mixture obtained in the step (2) to 12 by using 1mol/L sodium hydroxide, placing the mixture under 300W power for ultrasonic treatment for different time (0, 10, 20 and 30min), stirring the mixture for 1 hour at room temperature, and centrifuging the mixture for 10min at 10000r/min to obtain a supernatant;
(4) adjusting the pH value of the supernatant obtained in the step (3) to 4.5 by using 3mol/L hydrochloric acid, and centrifuging at the rotating speed of 12000r/min for 10min to collect protein precipitate;
(5) washing the protein precipitate obtained in the step (4) with double distilled water, adjusting the pH to 7 with 1mol/L NaOH, and then putting the protein precipitate into a dialysis bag of 8000Da and dialyzing the protein precipitate with deionized water for 12 hours to obtain a protein water solution;
(6) freeze-drying the protein aqueous solution obtained in the step (5) to obtain antarctic krill protein powder, and then mixing the protein powder and n-hexane according to a feed-liquid ratio of 1g:20mL for degreasing;
(7) and (4) centrifuging the mixed solution obtained in the step (6) at 6000r/min for 10min, and drying the obtained protein precipitate to obtain the euphausia superba protein.
In the embodiment, the euphausia superba protein is extracted by processing at 300W for different time (0, 10, 20 and 30min), and the influence of the processing for different ultrasonic time on the quality of the extracted protein is avoided. The particle size, foaming property, solubility and emulsifying property of the protein extracted from the euphausia superba treated by different ultrasonic time are improved in different ways. The particle size of the protein is one of the many factors that affect the functional properties of the protein, and smaller particle size proteins tend to have superior properties, as shown in figure 3: during the ultrasonic-assisted extraction process of the protein of the Antarctic krill, the particle size of the protein is gradually reduced along with the increase of the ultrasonic time. As shown in FIG. 4, the foaming ability and foaming stability of the protein obtained by ultrasonic-assisted extraction are improved, and the protein obtained by ultrasonic treatment for 20-30min has the best effect. Scanning electron microscope analysis (A is not ultrasonic; B is ultrasonic for 10 min; C is ultrasonic for 20 min; D is ultrasonic for 30min) is carried out on the protein extracted by different ultrasonic waves as shown in the figure 5, and the result shows that the structure of the euphausia superba protein is smaller and looser after ultrasonic treatment.
Example 4
(1) Preparing alkaline electrolyzed water: preparing 0.5 wt% sodium chloride aqueous solution, and electrolyzing in an electrolytic water device (model FW-200 of Tianye, Shenchuan county, Japan) for 10min to obtain alkaline electrolytic water (pH 12.31 + -0.21);
(2) and (3) mixing the unfrozen antarctic krill with alkaline electrolytic water according to the feed-liquid ratio of 1g: 5mL of the euphausia superba extract is mixed, the euphausia superba extract and deionized water are mixed according to the same proportion to serve as a reference, the pH value of the reference group is adjusted to 12 by using 1mol/L sodium hydroxide, and then the reference group is homogenized for 2min at the rotating speed of 800r/min to obtain a mixture;
(3) adjusting the pH value of the mixture obtained in the step (2) to 12 by using 1mol/L sodium hydroxide, performing ultrasonic treatment (0-30min) under the power of 300W, stirring for 2 hours at room temperature, and centrifuging for 10min at 8000r/min to obtain a supernatant;
(4) regulating the pH value of the supernatant obtained in the step (3) to 4.5 by using 3mol/L hydrochloric acid, and centrifuging at the rotating speed of 8000r/min for 10min to collect protein precipitate;
(5) washing the protein precipitate obtained in the step (4) with double distilled water, adjusting the pH to 7 with 1mol/L NaOH, and then putting the solution into a 10000Da dialysis bag to be dialyzed with deionized water for 12 hours to obtain a protein water solution;
(6) and (3) freeze-drying the protein aqueous solution obtained in the step (5) to obtain antarctic krill protein powder, and then mixing the protein powder with n-hexane according to a feed-liquid ratio of 1g: 5mL of the mixture is mixed for degreasing;
(7) and (4) centrifuging the mixed solution obtained in the step (6) at 6000r/min for 10min, and drying the obtained protein precipitate to obtain the euphausia superba protein.
The experiment on the oxidation of the extracted euphausia superba protein by using different ultrasonic time assisted alkaline electrolyzed water shows. Protein carbonyl is an early marker of various amino acids in the oxidative modification process of proteins, and the higher content of the protein carbonyl indicates the higher degree of oxidative damage of the proteins, and the protein carbonyl is a main marker for measuring the oxidative damage of the proteins. The results of this example are shown in fig. 6, the content of carbonyl in the alkaline electrolyzed water extracted antarctic krill protein is far lower than that of the protein extracted by deionized water, the ultrasound-assisted treatment increases protein exposure and slightly increases the carbonyl in the extracted protein, but the content of carbonyl is also far lower than that of the protein extracted by deionized water, so the reduction potential in the alkaline electrolyzed water during the protein extraction process of the method can effectively protect the protein from oxidation.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (10)
1. An extraction method of euphausia superba protein is characterized by comprising the following steps:
(1) mixing Antarctic krill with alkaline electrolyzed water, and homogenizing to obtain a mixture; the pH value of the alkaline electrolyzed water is 11.6-12.8, and the alkaline electrolyzed water is obtained by electrolysis of a sodium chloride aqueous solution;
(2) adjusting the pH value of the mixture obtained in the step (1) to 11.6-12.8, performing ultrasonic treatment, stirring, centrifuging and taking a supernatant;
(3) adjusting the pH of the supernatant obtained in the step (2) to 4-5, centrifuging, and collecting protein precipitate;
(4) washing the protein precipitate obtained in the step (3) with double distilled water, adjusting the pH to 7, and dialyzing to obtain a protein water solution;
(5) freeze-drying the protein aqueous solution obtained in the step (4) to obtain antarctic krill protein powder, and then mixing the protein powder with n-hexane for degreasing;
(6) and (5) centrifuging the mixed solution obtained in the step (5), and drying the obtained protein precipitate to obtain the euphausia superba protein.
2. The extraction method of euphausia superba protein according to claim 1, wherein the concentration of the aqueous sodium chloride solution in step (1) is 0.5-1.5 wt%, and the electrolysis time is 8-12 minutes.
3. The extraction method of euphausia superba protein according to claim 1, wherein the ratio of the euphausia superba and the alkaline electrolyzed water in step (1) is 1g: (5-10) mixing, wherein the homogenization condition is that the mixture is homogenized for 2-5min at the rotating speed of 500-800 r/min.
4. The extraction method of Euphausia superba protein as claimed in claim 1, wherein the ultrasonic power in step (2) is 300- "350W", the ultrasonic time is 20-30min, and the stirring condition is room temperature stirring for 1-2 hours.
5. The extraction method of Euphausia superba protein according to claim 1, wherein said pH adjustment in step (2) is performed using 0.8-1.2mol/L aqueous sodium hydroxide, and said pH adjustment in step (3) is performed using 2.8-3.2mol/L hydrochloric acid.
6. The extraction method of Euphausia superba protein as claimed in claim 1, wherein the centrifugation conditions in steps (2) and (3) are 8000-12000r/min for 10-15 min.
7. The extraction method of Euphausia superba protein as claimed in claim 1, wherein the dialysis conditions in step (4) are dialysis in 5000-12000Da dialysis bag for 10-24 hours.
8. The extraction method of euphausia superba protein according to claim 1, wherein the feed-to-liquid ratio of the protein powder and n-hexane in step (5) is 1g: (10-20) mL.
9. The extraction method of Euphausia superba protein as claimed in claim 1, wherein the centrifugation conditions in step (6) are 5000-8000r/min for 5-10 min.
10. An extraction method of antarctic krill protein, which is characterized by being prepared by the method of any one of claims 1 to 9.
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Inventor after: Zhao Yong Inventor after: Li Yufeng Inventor after: Wang Jingjing Inventor after: Liu Haiquan Inventor after: Wu Yi Inventor after: Wang Ronghan Inventor before: Zhao Yong Inventor before: Li Yufeng Inventor before: Wang Jingjing Inventor before: Liu Haiquan Inventor before: Wu Yi Inventor before: Wang Ronghan |
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Application publication date: 20200428 |