CN112480241A

CN112480241A - Method for extracting phycocyanin from fresh spirulina harvested in high-salt environment

Info

Publication number: CN112480241A
Application number: CN202011531300.4A
Authority: CN
Inventors: 苏勇宁; 李玉国
Original assignee: Inner Mongolia Rejuve Biotech Co ltd
Current assignee: Inner Mongolia Rejuve Biotech Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-03-12

Abstract

The invention discloses a method for extracting phycocyanin from fresh spirulina harvested in a high-salt environment, which takes the fresh spirulina as a raw material, fully washes and desalts the fresh spirulina, puts the fresh spirulina in soft water with 2-3 times of fresh spirulina volume for fully soaking and permeating and impacting for 2-3 hours, adds 0.2-1.0mol/L soluble carboxylate or sulfonate into the soaking solution, fully stirs and dissolves the cell for 5-6 hours, stirs and cuts the solubilizing solution for 1-2 hours by using a high-speed dispersion machine with 1000-plus 1500rmp/min to fully break spirulina cells; and separating the wall-broken liquid by a centrifuge to remove residues, concentrating by an ultrafiltration membrane and drying to obtain the high-purity phycocyanin. The phycocyanin protein is extracted from the fresh algae, so that the advantage of saving the drying cost from the algae mud to the algae powder is achieved; the extraction process is simple, and compared with other methods, the method has the advantages of less salt components, low cost and less pollution emission; the extraction time can be shortened by adopting a centrifugal deslagging mode.

Description

Method for extracting phycocyanin from fresh spirulina harvested in high-salt environment

Technical Field

The invention relates to an extraction process for extracting phycocyanin from fresh spirulina, belonging to the technical field of natural pigment extraction.

Background

The spirulina is a prokaryotic organism capable of photosynthesis, can grow well in a high-salt and high-alkaline environment, has excellent economic value, and has a yield of one thousand tons in China after development of nearly 30 years. Spirulina is rich in phycocyanin, a natural blue pigment protein, the content of which is up to 25% of the dry cell weight, and phycocyanin has been listed in food additives.

The spirulina belongs to gram-negative bacteria, the cell wall has three layers, the outer layer mainly comprises protein and lipopolysaccharide, the middle layer mainly comprises peptidoglycan, the inner layer mainly comprises phospholipid, the content of peptidoglycan in the cell wall of the gram-negative bacteria is low, the cell wall is relatively thin, and phycocyanin protein can be extracted by easily damaging the cell wall. The loss of phycocyanin in spirulina after spray drying exceeds 10% in a high-temperature link, and the phycocyanin can be extracted by directly utilizing the phycomud, so that the extraction rate of phycocyanin can be increased, and the stability of phycocyanin can be improved. Phycocyanin protein is not resistant to high temperature in liquid state, and is easy to lose color when the temperature exceeds 40 ℃, and the extraction process needs to be kept in a low temperature state. At present, the extraction process of phycocyanin mainly focuses on a wall breaking method for extracting phycocyanin from spirulina by a freeze thawing method, a chemical reagent method, a mechanical wall breaking method or a combination method thereof. However, most of the extraction methods focus on the extraction process of fresh spirulina or spirulina powder in low-salt environment, and less methods suitable for industrial production of phycocyanin from fresh spirulina cultured in high-salt environment.

In order to improve the extraction efficiency of phycocyanin protein and produce phycocyanin with good stability and high purity, the invention provides a method for extracting phycocyanin from fresh spirulina in a high-salt environment by comprehensively utilizing the principles of a chemical impact method, a solubilization method and mechanical wall breaking.

Disclosure of Invention

The invention aims to provide a method for extracting phycocyanin from fresh spirulina harvested in a high-salt environment, so as to eliminate technical obstacles for extracting phycocyanin from spirulina cultured in a high-salt and alkali environment by using trona in inner Mongolia areas.

In order to achieve the purpose, the invention adopts the following method for extraction, fresh spirulina is taken as a raw material, the fresh spirulina is fully washed and desalted, then the fresh spirulina is placed in soft water with the volume 2 to 3 times of that of the fresh spirulina for full soaking and osmotic shock for 2 to 3 hours, 0.2 to 1.0mol/L soluble carboxylate or sulfonate is added into a soaking solution, the full stirring and dissolution are carried out for 5 to 6 hours for solubilizing cells, and the solubilizing solution is stirred and cut for 1 to 2 hours by a high-speed dispersion machine with the volume of 1000 plus 1500rmp/min for full crushing of spirulina cells; and separating the wall-broken liquid by a centrifuge to remove residues, concentrating by an ultrafiltration membrane and drying to obtain the high-purity phycocyanin. The method comprises the following specific steps:

1. taking fresh dehydrated spirulina mud cultured in a high-salt environment as an extraction raw material, wherein the salinity of a culture solution of spirulina is more than 10g/L, the spirulina mud is fully washed, desalted and dehydrated, the water content of the spirulina mud is less than 90 percent, the spirulina mud is placed in soft water with the volume of 2 to 3 times of that of the spirulina mud and fully and uniformly stirred to obtain spirulina mud suspension, and the spirulina mud is kept stand for 2 to 3 hours;

2. after standing, adding 0.2-1.0mol/L soluble carboxylate or sulfonate into the soak solution, fully stirring and dissolving to solubilize the cells for 5-6 hours;

3. stirring and cutting the solubilization solution for 1-2 hours by using a high-speed dispersion machine of 1000-1500rmp/min to fully break the spirulina cells;

4. separating the wall-broken liquid by a centrifuge to remove residues, concentrating by an ultrafiltration membrane, and drying to obtain high-purity phycocyanin, wherein the residue removal centrifugation is divided into two stages, the first stage uses the centrifuge with a separation factor of about 6000 to remove large-particle algae residues, the second stage uses the centrifuge with a separation factor of not less than 12000 to remove small-particle impurities, the aperture of the ultrafiltration concentration membrane is 10000D, the concentration is not less than 0.6 when the OD618 is concentrated, and the drying is preferably freeze drying, and the next stage is spray drying.

The phycocyanin protein is extracted from the fresh algae, so that the advantage of saving the drying cost from the algae mud to the algae powder is achieved; the extraction process is simple, and compared with other methods, the method has the advantages of less salt components, low cost and less pollution emission; the extraction time can be shortened by adopting a centrifugal deslagging mode.

Description of the drawings:

FIG. 1 shows high purity phycocyanin obtained by the present invention.

The specific implementation mode is as follows:

example 1:

1. taking fresh dehydrated spirulina mud cultured in a high-salt environment (the salinity of a culture solution is 13.2g/L) as an extraction raw material, fully washing, desalting and dehydrating the spirulina mud, weighing 1980g of the spirulina mud with the water content of 86.2 percent, putting the spirulina mud in 3 times of volume of soft water, fully and uniformly stirring to obtain spirulina mud suspension, and standing for 3 hours;

2. after standing, adding 0.6mol/L sodium citrate into the soak solution, fully stirring and dissolving to solubilize the cells for 6 hours;

3. stirring and cutting the solubilized solution for 1.5 hours by using a high-speed dispersion machine of 1500rmp/min to fully break the spirulina cells;

4. and (2) removing large-particle algae residues from the wall-broken liquid by using a centrifuge with a separation factor of 6000 in the first stage, removing small-particle impurities from the wall-broken liquid by using a centrifuge with a separation factor of 15000 in the second stage, concentrating the wall-broken liquid by using an ultrafiltration membrane with an ultrafiltration membrane pore diameter of 10000D until the OD618 value is 0.75, precooling the concentrated liquid for 10 hours at the temperature of-20 ℃, and then treating and drying the concentrated liquid by using a vacuum freeze dryer to obtain 43.5g of high-purity phycocyanin shown in figure 1, wherein the purity OD618/OD280 of the phycocyanin is 0.362.

Example 2:

1. taking fresh dehydrated spirulina mud cultured in a high-salt environment (the salinity of a culture solution is 12.4g/L) as an extraction raw material, fully washing, desalting and dehydrating the spirulina mud, weighing 15.68kg of spirulina mud with the water content of 86.8%, putting the spirulina mud in soft water with the volume of 3 times of that of the spirulina mud, fully and uniformly stirring to obtain spirulina mud suspension, and standing for 3 hours;

2. after standing, adding 0.5mol/L sodium citrate into the soak solution, fully stirring and dissolving to solubilize the cells for 6 hours;

4. and (3) passing the wall breaking liquid through two centrifuges, removing large-particle algae residues by using a centrifuge with a separation factor of 6000 at the first stage, removing small-particle impurities by using a centrifuge with a separation factor of 15000 at the second stage, concentrating the concentrated solution to an OD618 value of 0.85 by using an ultrafiltration membrane with an ultrafiltration membrane pore diameter of 10000D, and spray-drying the concentrated solution at an air inlet temperature of 190 ℃ to obtain 310.5g of high-purity phycocyanin shown in figure 1, wherein the purity OD618/OD280 is 0.358.

Claims

1. The method for extracting phycocyanin from fresh spirulina harvested in high-salt environment is characterized by comprising the following steps of:

(1) taking fresh dehydrated spirulina mud cultured in a high-salt environment as an extraction raw material, wherein the salinity of a culture solution of spirulina is more than 10g/L, the spirulina mud is fully washed and dehydrated, the water content of the spirulina mud is less than 90%, placing the spirulina mud in soft water with the volume of 2-3 times of that of the spirulina mud, fully and uniformly stirring to obtain spirulina mud suspension, and standing for 2-3 hours;

(1) after standing, adding 0.2-1.0mol/L soluble carboxylate or sulfonate into the soak solution, fully stirring and dissolving to solubilize the cells for 5-6 hours;

(2) stirring and cutting the solubilization solution for 1-2 hours by using a high-speed dispersion machine of 1000-1500rmp/min to fully break the spirulina cells;

(3) separating the wall-broken liquid by a centrifuge, removing residues, concentrating by an ultrafiltration membrane, and drying to obtain high-purity phycocyanin.

2. The method for extracting phycocyanin from fresh spirulina that is suitable for high salinity environment as claimed in claim 1, wherein the molecule of the soluble carboxylate or sulfonate should contain at least two carboxyl or sulfonic acid groups.

3. The method for extracting phycocyanin from fresh spirulina that is suitable for high salinity environment as claimed in claim 1, wherein the temperature of the liquid is controlled to be less than 10 ℃ during the whole extraction process.

4. The method of claim 1, wherein the step of removing the residues and centrifuging the residues is performed in two stages, wherein the first stage is performed by removing large-particle algae residues with a centrifuge having a separation factor of about 6000, and the second stage is performed by removing small-particle impurities with a centrifuge having a separation factor of not less than 12000.

5. The method for extracting phycocyanin from fresh spirulina harvested in high salinity environment as claimed in claim 1, wherein the best drying method is vacuum freeze drying, and the next step is spray drying.