CN115109146A

CN115109146A - Algae protein extracted from algae and preparation method thereof

Info

Publication number: CN115109146A
Application number: CN202210931366.5A
Authority: CN
Inventors: 张天; 王力; 边朝阳
Original assignee: Beijing Yueda Biotechnology Co ltd
Current assignee: Beijing Yueda Biotechnology Co ltd
Priority date: 2022-08-04
Filing date: 2022-08-04
Publication date: 2022-09-27

Abstract

The invention relates to an algae protein extracted from algae and a preparation method thereof, belonging to the technical field of plant protein extraction. The method comprises the following steps: (1) enzymatic wall breaking of algae powder; (2) precipitating the wall-broken solution with anhydrous calcium chloride, and filtering to obtain filtrate; (3) filtering the clear liquid, performing ultrafiltration concentration to 20% -25% of the original volume to obtain concentrated solution, spraying powder and drying to obtain the algae protein. The invention utilizes the cellulase method to break the wall, has high wall breaking efficiency which reaches more than 99 percent, low cost, mild reaction condition and easy operation; in addition, the method removes impurities such as chlorophyll and the like by using the precipitate of anhydrous calcium chloride, removes impurity proteins by an ultrafiltration method, and finally obtains high-purity algal protein, wherein the purity of the phycocyanin is more than 30 percent, and the yield is more than 99 percent; the purity of phycoerythrin is more than 40%, the yield is more than 99%, and the fishy smell of spirulina can be removed.

Description

Algae protein extracted from algae and preparation method thereof

Technical Field

The invention relates to an algae protein extracted from algae and a preparation method thereof, belonging to the technical field of plant protein extraction.

Background

The spirulina is blue algae in the microalgae, has the advantages of high protein, low fat, low sugar and low cholesterol, is rich in nutrients such as vitamins, proteins, fat, carbohydrates, minerals, trace elements and the like, has balanced proportion of the nutrients, has very good edible health-care function, and is a natural food. Is praised as a miniature nutrition bank and the most ideal and excellent food for human. And simultaneously has various pharmacological effects. Such as reducing blood fat, resisting oxidation, resisting infection, resisting radiation, resisting aging, enhancing immunity, etc.

The phycocyanin is an active protein separated and purified from blue algae, and a rare frightening pigment protein which is a natural edible pigment allowed to be used by the country, and more importantly, researches show that the phycocyanin can obviously relieve the damage to normal white blood cells of a human body during tumor radiotherapy and chemotherapy, stimulate the colony formation of red blood cells and platelets, improve the immunity and protect liver tissues. It has unique fluorescent property and good biological efficacy. Has the same properties as protein. The product is blue granules or powder. Dissolved in water. Insoluble in alcohol and oil. Organic solvents such as ethanol can cause irreversible denaturation of phycocyanin. The aqueous solution was vivid blue in color. Phycocyanin is a conjugated protein formed by covalently linking apoprotein and phycocyanin through one or two thioether bonds. Each phycocyanin monomer is composed of alpha and beta subunits, and its monomer (alpha beta) has a molecular weight of about 40Kd, is present as a trisome (alpha beta) at pH6.5-8.0, and is present as a hexame (alpha beta) at pH4.5-6.0, and has a molecular weight of about 230 Kd. Whether the compound exists in a monomer, a trisomy or a hexamer form, the compound shows beautiful sapphire color under visible light, has bright color and is determined to be quite stable under various physical and chemical factors. The high-purity phycocyanin has strong fluorescence characteristics, and can be used for preparing fluorescent probes for scientific detection and research.

Phycoerythrin is a pigment protein present in red algae, blue algae and crypthecodinium, and open-loop tetrapyrrole phycoerythrin as a pigment part is covalently bonded to the protein. The prosthetic group is a chain formed by pyrrole rings, does not contain metal in the molecule and is combined with protein. The subunit composition of the protein is (. alpha. -beta.) 6. gamma., each of the. alpha. -and. beta. -subunits is about 20kD, each of the. gamma. -subunits is about 30kD, and its molecular weight is 240 KD. The phycoerythrin has maximum absorption in the visible light area of 565nm and can produce strong fluorescence, and the fluorescence intensity is 30-100 times of that of fluorescein. Has good light absorption performance and high quantum yield, has wide excitation and emission range in visible spectrum region, and has fluorescence emission peak of about 578 nm. Has a certain degree of stability, can be applied to food and cosmetics, and can also be used as a fluorescent pigment for antibody markers in immunology, so that the fluorescent pigment can be applied to clinical diagnosis and cytobiochemical research. The fluorescent probe can be conveniently combined with biotin, avidin and various monoclonal antibodies by a conventional labeling method to prepare the fluorescent probe for clinical diagnosis and bioengineering technologies such as immunodetection, fluorescence microscopy, flow cytometry fluorescence measurement and the like.

Presently phycoerythrin is mostly isolated from the large fronds of red algae. The laver is the most common one, and the phycobiliprotein is contained in the laver in a large amount, and accounts for about 4% of the dry weight of the laver. Phycobiliprotein is the general name of phycoerythrin, phycocyanin and allophycocyanin, and phycoerythrin is the main component in laver.

The traditional phycocyanin or phycoerythrin separation technology generally adopts the methods of high-pressure homogenization, repeated freezing and thawing, ultrasound and the like to break cells for extraction, and then ammonium persulfate protein precipitation and chromatographic chromatography are carried out for purification. Inconvenient operation, large energy consumption, large environmental pollution, difficult treatment of waste water and difficult large-scale production.

Disclosure of Invention

The invention aims to provide algae protein extracted from algae and a preparation method thereof.

The technical scheme of the invention is as follows:

a method for extracting protein from algae comprises the following steps:

(1) breaking cell wall of algae powder by enzyme method, wherein water accounts for 10-15 times of the mass of the algae powder, and cellulase accounts for 0.1-0.15 time of the mass of the algae powder; the reaction temperature is 25-35 ℃, the reaction time is 15-20h, and after the reaction is finished, plate-and-frame filtration is carried out to obtain a wall-breaking liquid;

(2) precipitating the wall-broken solution with anhydrous calcium chloride, and filtering to obtain clear filtrate; adding anhydrous calcium chloride in an amount of 0.5-2% (mass fraction) of the wall-broken solution, stirring, standing for 15-20 hr, and filtering to obtain filtrate;

(3) filtering the clear liquid, performing ultrafiltration concentration to 20% -25% of the original volume to obtain concentrated solution, spraying powder and drying to obtain the algae protein.

Preferably, the algae powder in the step (1) takes spirulina or laver as a raw material; when the spirulina is used as the raw material of the algae powder, water accounts for 10 times of the mass of the algae powder, and cellulose accounts for 0.1 time of the mass of the algae powder; the reaction temperature is 25 ℃, and the reaction time is 20 hours; when the laver is used as the raw material of the algae powder, the water accounts for 10 to 15 times of the weight of the laver powder, and the cellulose accounts for 0.1 to 0.15 time of the weight of the laver powder; the reaction temperature is 25-35 ℃, and the reaction time is 15-20 h.

Preferably, the addition amount of the anhydrous calcium chloride in the step (2) is 1% (mass fraction) of the wall breaking liquid, and the anhydrous calcium chloride is fully and uniformly stirred and then is kept stand for 15-20 h; preferably, the filtration is performed by using a disk centrifuge or plate-and-frame filtration.

Further, the present invention also includes algal proteins obtained by the above method.

Compared with the prior art, the invention has the following advantages:

(1) the invention utilizes the cellulase method to break the wall, has high wall breaking efficiency which is more than 99 percent (the phycocyanin can not be detected in the slag after the wall breaking), has low cost, mild reaction condition and easy operation.

(2) Removing impurities such as chlorophyll and the like by using the precipitate of anhydrous calcium chloride, and removing impurity proteins by using an ultrafiltration method to finally obtain high-purity algal protein, wherein the purity of the phycocyanin is more than 30 percent, and the yield is more than 99 percent; the purity of phycoerythrin is more than 40%, the yield is more than 99%, and the fishy smell of spirulina can be removed.

Detailed Description

The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

Example 1: method for extracting phycocyanin from spirulina

(1) Taking 100g of spirulina powder, adding 1000mL of water, stirring uniformly, adding 10g of cellulase respectively, stirring for 30min, and stopping stirring after uniform stirring; standing at room temperature for 18h, and filtering with plate frame to obtain wall-broken solution 920 g.

(2) 9.2g of anhydrous calcium chloride is added, after uniform stirring, the mixture is kept stand for 20 hours, and then filtered by a plate-and-frame filter to obtain 902g of filtered clear liquid.

(3) And (3) ultrafiltering, concentrating and removing impurities from the filtered clear liquid by using a 100kd ultrafiltration membrane to obtain 215g of concentrated solution, spraying the concentrated solution and drying to obtain 38g of phycocyanin powder with the content of 31% and the yield of 99%.

Example 2A method for extracting phycoerythrin from Spirulina

The method comprises the steps of taking laver, pulverizing, weighing 100g, adding 1000mL of water, stirring uniformly, adding 10g of cellulase and 10g of beta-glucanase respectively, stirring for 30min, stopping stirring after the mixture is uniform, standing for 18h at room temperature, filtering to obtain 918g of wall breaking liquid, adding 9.2g of anhydrous calcium chloride, stirring uniformly, standing for 20h, and filtering to obtain 895g of filtered clear liquid. And (3) ultrafiltering, concentrating and removing impurities from the filtered clear liquid by using a 100kd ultrafiltration membrane to obtain 225g of concentrated solution, and freeze-drying to obtain 9.5g of phycoerythrin powder, wherein the content of phycoerythrin is 41 percent, and the yield is 99 percent.

Example 3A method for extracting phycoerythrin from Spirulina

The method comprises the steps of collecting laver, powdering, weighing 100g, adding 800mL of water, stirring uniformly, adding 15g of cellulase respectively, stirring for 30min, stopping stirring after uniform stirring, standing at room temperature for 16h, filtering to obtain wall-broken liquid 918g, adding 9.0g of anhydrous calcium chloride, stirring uniformly, standing for 20h, and filtering to obtain filtered clear liquid 902 g. And (3) ultrafiltering, concentrating and removing impurities from the filtered clear liquid by using a 100kd ultrafiltration membrane to obtain 225g of concentrated solution, and freeze-drying to obtain 9.2g of the phycoerythrin powder, wherein the content of the phycoerythrin is 42 percent, and the yield is 99 percent.

Example 4: method for extracting phycocyanin from spirulina

Taking 10kg of spirulina powder, adding 90kg of water, uniformly stirring, respectively adding 1.5kg of cellulase, uniformly stirring for 1h, stopping stirring, standing at room temperature for 16h, filtering to obtain 91.75kg of wall-breaking liquid, adding 10.0kg of anhydrous calcium chloride, uniformly stirring, standing for 20h, and filtering to obtain 88.54kg of filtrate. And (3) ultrafiltering, concentrating and removing impurities from the filtered clear liquid by using a 100kd ultrafiltration membrane to obtain 24.26kg of concentrated solution, and freeze-drying to obtain 3.65kg of phycocyanin powder with the phycocyanin content of 31.5% and the yield of 99%.

Claims

1. A method for extracting proteins from algae, the method comprising the steps of:

(2) precipitating the wall-broken solution with anhydrous calcium chloride, and filtering to obtain filtrate; adding anhydrous calcium chloride in an amount of 0.5-2% (mass fraction) of the wall-broken solution, stirring, standing for 15-20 hr, and filtering to obtain filtrate;

2. The method as claimed in claim 1, wherein the algae powder of step (1) is prepared from spirulina or laver.

3. The method of claim 2, wherein when the algae powder is spirulina, the amount of water is 10 times the amount of the algae powder, and the amount of cellulose is 0.1 times the amount of the algae powder; the reaction temperature is 25 ℃, and the reaction time is 20 h.

4. The method of extracting protein from algae according to claim 2, wherein when the algae powder is laver, water is 10-15 times the weight of the laver powder, and the cellulase is 0.1-0.15 times the weight of the laver powder; the reaction temperature is 25-35 ℃, and the reaction time is 15-20 h.

5. The method of claim 1, wherein the amount of anhydrous calcium chloride added in step (2) is 1% (by weight) of the wall-breaking liquid, and the mixture is stirred well and then allowed to stand for 15-20 h.

6. The method for extracting protein from algae according to claim 1, wherein the filtering in step (2) is performed by a disk centrifuge or plate-and-frame filtering.

7. Algal proteins obtained by the method for extracting proteins from algae according to any one of claims 1 to 6.