CN110256542A

CN110256542A - A kind of preparation method of red algae phycoerythrin

Info

Publication number: CN110256542A
Application number: CN201910475267.9A
Authority: CN
Inventors: 江晓路; 王纯
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-06-03
Filing date: 2019-06-03
Publication date: 2019-09-20

Abstract

The present invention provides a kind of easy to operate, low in cost and suitable industrialized production, the method of PE is extracted from red algae, the method that the present invention selects β-agarase and cellulase to extract phycoerythrin in marine red alga, the time is saved compared with traditional technology, is reduced costs, greatly simplify operation, the rate of recovery is high, and purity is high provides a kind of method of simplicity for the industrialized production of phycoerythrin.

Description

A kind of preparation method of red algae phycoerythrin

Technical field

The invention belongs to a kind of field of biotechnology, are related to a kind of side that phycoerythrin in red algae is extracted using agarase Method.

Background technique

Red algae refers to the algae of Rhodophyta.In red algae, there are some edible types full of nutrition, delicious flavour such as purple Dish, Eucheuma, gloiopeltis etc.；There are also some important economic species, for extracting agar-agar and carragheen, such as agar, fragrant plant mentioned in ancient texts, wrinkle The plants such as wave Irish moss, Eucheuma, majority are many cells, minority be it is single celled, which only has Rhodophyceae, there are about 760 categories, More than 4410.Rhodophyceae is divided to two subclasses: seaweed subclass and true Rhodophyceae again；Red algae is rich in bioactive substance (albumen because of it Matter, polyunsaturated fatty acid and polysaccharide) and there is good bioactivity (anti-oxidant, anti-inflammatory, antibacterial and antitumor) and well-known, Dynamics and depth also very little that China develops and uses red algae at present are largely only limitted to simply edible.Phycoerythrin (PE) Be it is important in red algae catch photochromic fibroin, molecular weight has relatively strong in 240 kDa or so in 490~570 nm visible regions Light absorption, α and β subunit is the basic building block of PE, and a α subunit and a β subunit constitute a monomer, and PE is usually to contain The tripolymer or six aggressiveness of 3 or 6 this monomers, the form that is primarily present are (α β)₆γ, state of aggregation is higher, and PE's catches luminous energy Power and Light energy transfer ability are stronger.PE has high water soluble, is used for foods and cosmetics industry frequently as colorant, because of its tool There is fluorescent characteristic, can be used as fluorescent marker, tracer, immune fluorescent probe etc. is used for biological medicine and immunological investigation.According to report Road PE has multiple biological activities, such as in addition to this antitumor, anti-diabetic, Immune-enhancing effect and anti-hypertension are also developed At a kind of photosensitizer, it to be used for photodynamic therapy for cancer.PE is intracellular compound, since red algae cell wall is mainly by polysaccharide (agar, cellulose, xylan etc.) composition, limits the release of PE.Extracting mode traditional at present has physical disruption methods (ultrasound It is broken, liquid nitrogen grinding, multigelation), chemical osmosis (cell swelling method, CaCl₂Solution extraction method), aqueous two-phase system, Flocculence etc., since these modes are at high cost, and time-consuming, and yield is low etc., reasons limit its industrialized production.Enzymatic isolation method is a kind of The effective alternative for promoting alga cells disorganization release compound, uses early in Joubert in 2008 and Fleurence Zytase and cellulase are studied, it was confirmed that enzyme recycles the feasibility of PE, and publication number CN 101617784A is also disclosed A method of phycobniliprotein in seaweed being extracted using cellulase and pectase, but currently without using agarase assisted extraction The report of PE occurs.It is well known that red algae cell wall contains a large amount of agar-agar, this is also the original that red algae extracts source as agar-agar Agar polysaccharide in cell wall can be degraded into oligosaccharides by cause, agarase, to more fully discharge PE.

The present invention improves the yield of PE by the way that β-agarase and cellulase, ammonium sulfate precipitation, the technologies such as ultrafiltration is added And purity, reduce extraction cost, reduce extraction time, and it is freeze-dried after obtained phycoerythrin freeze-dried powder can be at low temperature Long-term preservation is suitable for industrialized production, can promote industrial upgrading.

Summary of the invention

The present invention provides a kind of easy to operate, low in cost and suitable industrialized production, the method for extraction PE from red algae, Particular content is thoroughly to be crushed with pulverizer by after red algae drying, cross 80 meshes, 0.1 M of red algae powder and pH 6.8~7.2 Phosphate buffer is mixed well in 1:25~1:35 ratio, obtains the 0.1M phosphoric acid of suspension or fresh frond and pH 6.8~7.2 Buffer is homogenized by 1:2~3, and 0.001%~1% β-agarase and 0.001%~1% fibre of mass concentration are added under specified conditions Plain enzyme is tieed up, 4~6 h of enzymatic hydrolysis at 25~35 DEG C is placed in, obtains shell-broken liquid.Obtained shell-broken liquid is freezed in 8000 r/min 10 min are centrifuged, supernatant is obtained, the ammonium sulfate that saturation degree is 30%~60% is added to supernatant, stands overnight at low temperature, Again with 8000 r/min refrigerated centrifuge, 10 min, the 0.1M phosphate buffer for taking precipitating that pH 6.8~7.2 is added redissolves.It redissolves Protein solution afterwards is concentrated by ultrafiltration, and the molecular cut off of ultrafiltration membrane used is 50000 Da.Ultrafiltration desalting efficiency is than dialysis Want high, the also liquid volume of reduction while realizing quick desalination is conducive to subsequent freeze-drying, is more suitable for industrial metaplasia It produces.

The technical advantage of invention: the method that the present invention selects β-agarase and cellulase to extract PE in marine red alga, with Traditional technology is reduced costs compared to the time is saved, and greatly simplifies operation, and the rate of recovery is high, purity is high, and have passed through experiment Room verifying, provides a kind of method of simplicity for the industrialized production of PE.

Specific embodiment

Embodiment 1.

(1) it pre-processes: the seaweed after drying is crushed with pulverizer, it is spare to obtain seaweed dry powder for 80 meshes excessively.

(2) the 0.1M phosphate buffer of seaweed powder and pH 6.8~7.2 in step (1) is sufficiently mixed in 1:30 ratio It is even, it is spare to obtain mixed liquor.

(3) β-agarase and 0.02% of mass concentration 0.02% is sequentially added into cell suspension obtained in step (2) Cellulase stirs at low speed 5 h of lower 30 DEG C of enzymatic hydrolysis, obtains shell-broken liquid.

(4) the obtained seaweed shell-broken liquid of step (3) is obtained into supernatant in 8000 r/min refrigerated centrifuge, 10 min.

(5) ammonium sulfate that saturation degree is 50% is added in the supernatant in step (4), stands overnight at low temperature, then with 8000 r/min refrigerated centrifuge, 10 min, the 0.1M phosphate buffer for taking precipitating that pH 6.8~7.2 is added redissolve.

(6) protein solution after redissolving in step (5) is concentrated by ultrafiltration, the molecular cut off of ultrafiltration membrane used is 50000 Da.PE purity (A₅₆₀/A₂₈₀) reach 1.2, yield is 0.83%(relative to seaweed dry weight).

(7) after ultrafiltration, solution is freeze-dried to obtain PE powder.The PE powder of freeze-drying can long-term preservation at low temperature, Activity keeps good, is conducive to industrialized production and product development.

Embodiment 2.

(1) it pre-processes: fresh frond being washed with water completely, draining is stand-by.

(2) step (1) fresh frond and the 0.1M phosphate buffer of pH 6.8~7.2 is spare by the homogenate of 1:2~3.

(3) β-agarase and 0.01% of mass concentration 0.01% is sequentially added into cell suspension obtained in step (2) Cellulase stirs at low speed 5 h of lower 30 DEG C of enzymatic hydrolysis, obtains shell-broken liquid.

(4) the obtained seaweed shell-broken liquid of step (3) is obtained into supernatant in 8000 r/min low-temperature centrifugation, 10 min.

(5) sulfuric acid that saturation degree is 50% is added by being concentrated under reduced pressure into the 1/5 of original volume in the supernatant in step (4) Ammonium is stood overnight at low temperature, then with 8000 r/min refrigerated centrifuge, 10 min, takes precipitating that the 0.1M of pH 6.8~7.2 is added Phosphate buffer redissolves.

(7) after ultrafiltration, solution is freeze-dried to obtain phycoerythrin powder.The phycoerythrin powder of freeze-drying can be in low temperature Lower long-term preservation, the technology are conducive to industrialized production and keep Product Activity.

Claims

1. the dry frond of red algae or fresh frond addition β-agarase and cellulose degraded in buffer after homogenate is smashed obtain To shell-broken liquid；Ammonium sulfate is added after carrying out centrifugal solid-liquid separation in obtained substance, stands overnight at low temperature, then with 8000 10 min of r/min refrigerated centrifuge, the 0.1M phosphate buffer that pH 6.8~7.2 is added in precipitating redissolve, the protein solution after redissolution It is concentrated by ultrafiltration, the molecular cut off of ultrafiltration membrane used is 50000 Da.

2. enzyme used in enzymatic hydrolysis according to claim 1 is that β-agarase and cellulase compound according to a certain percentage.

3. the use scope of enzyme used in enzymatic hydrolysis according to claim 1 is 0.001~1% β-agarase and 0.001~1% Cellulase.