CN114702561B - Method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria - Google Patents
Method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria Download PDFInfo
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Abstract
The invention relates to a method for comprehensively extracting phycobiliprotein and carrageenan from weak and thin-feather vegetables, which comprises the following steps of (1) crushing the weak and thin-feather vegetables and repeatedly freezing and thawing broken cells to obtain a phycobiliprotein crude extract; (2) Removing impurities from the crude extract by salting out, dialysis and other modes; (3) Purifying by phenyl-agarose puffing column and dextran G-150 gel chromatography column to obtain analytically pure phycoerythrin; (4) Adding potassium hydroxide with corresponding concentration into the algae residue separated in the step (1) according to a certain feed-liquid ratio for soaking; (5) Washing the algae residue with acid, bleaching, and washing with deionized water until the pH value is 7-8; (6) Adding deionized water with a certain proportion into the cleaned algae residue in the step (5), and heating and extracting in a water bath; (7) Filtering while hot to obtain filtrate, cooling, freezing and thawing, removing water, and oven drying to constant weight to obtain carrageenan. The invention takes the fine and weak plumes as raw materials, improves the economic value of the fine and weak plumes, and realizes the high-valued utilization of the fine and weak plumes.
Description
Technical Field
The invention relates to the technical field of seaweed biochemical preparation, in particular to a method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria.
Background
The genus Solieria (Solieria tenuis) is of the phylum Rhodophyta (Rhodophyta), rhodophyceae (Rhodophyceae), europe (Florideae), cunninghamiales (Gigartinales), solieriaceae (Solieriaceae), solieria (Solieria). The fine and weak plumes are mainly distributed in the rock or stone biogas of the Zhongchao zone in coastal areas of China and Japan and are often used as food cold dishes or cultivation feeds. At present, the functional active substances in the weak plumes are lack of systematic research.
Phycobiliprotein is a natural water-soluble protein with fluorescence, and is divided into phycoerythrin, phycocyanin, allophycocyanin and phycoerythrin. Phycobiliprotein is classified into food grade, medicine grade and reagent grade according to purity, and the value is gradually increased along with the improvement of purity. Phycobiliprotein can be used as natural colorant in food and chemical fields, and can also be used as antioxidant, fluorescent labeling agent, tumor inhibitor and photosensitizer in fields of medical care, biological detection, photodynamic therapy, etc. The phycobiliprotein has wide application range and high economic value, and the high-purity phycoerythrin in China still depends on import at present and is high in price, so that the extraction source of the phycoerythrin is expanded, the high-purity phycoerythrin is extracted in an optimized scale, and the high-value utilization of seaweed is facilitated.
The weak Solieria is a kind of Solieriaceae and contains carrageenan. The substance is a hydrophilic colloid, which is formed by alternately connecting sulfated or non-sulfated galactose and 3, 6-dehydrated galactose through alpha-1, 3 glycosidic bonds and beta-1, 4 bonds, and can be classified into seven types of carrageenan of kappa-type, iota-type, lambda-type, gamma-type, v-type, zeta-type and mu-type according to the bonding positions of sulfuric acid bonds, wherein kappa-type, iota-type and lambda-type carrageenan are more commonly used. Compared with agar, the carrageenan has stable performance and wide application. Can be used in food industry and daily chemical industryThickening agent、Gelling agentCan be used for jelly, canned food, sausage product, etc.; as a clarifying flocculant for beer, wine and the like; as a means ofSuspending agent、Emulsifying agentAndstabilizing agentUsed for products such as toothpaste, paint, pesticide spray and the like; in the aspects of bioengineering and biological medicine, can replace agar for culturingThe nutrient medium can be used as soluble dietary fiber for regulating intestinal balance. In addition to the shortage of carrageenan, the price of carrageenan is gradually increased in recent years, and the price of seaweed for extracting carrageenan is increased, at present, the carrageenan is mainly eucheuma, agar and the like, and the carrageenan is produced in a large quantity, so that part of algae resources are tense, and therefore, the fine and weak red plumes are used as new extraction sources of the carrageenan, so that the extraction sources of the carrageenan are expanded, the tense of part of extracted algae resources is relieved, and the economic value of the fine and weak red plumes is also improved.
At present, a certain bioactive substance is extracted singly, and few documents for simultaneously extracting a plurality of bioactive substances exist. The method for comprehensively extracting phycobiliprotein and carrageenan from the thin and weak plumes is not provided in the prior art, so that the method takes the thin and weak plumes as raw materials, comprehensively extracts phycoerythrin and carrageenan, enriches the raw material sources of the phycoerythrin and the carrageenan, is beneficial to realizing the high-value utilization of the thin and weak plumes and promotes the development and research of the thin and weak plumes. Before the subject group, phycoerythrin and agar are extracted by taking asparagus as raw materials, and only phycoerythrin with purity of 2.95 is obtained. Compared with the authorized method for extracting the polysaccharide from the algae by using alkali extraction, enzymolysis and alcohol precipitation, the method for extracting the polysaccharide has the advantages of simple steps, reduced extraction time, lower extraction cost and contribution to large-scale extraction.
Disclosure of Invention
The invention aims at solving the problems and provides a method for comprehensively extracting phycobiliprotein and carrageenan from weak plumes, which takes the weak plumes as raw materials to obtain reagent-grade pure phycoerythrin, and utilizes the algae residues after extracting the phycobiliprotein to extract the carrageenan, so that two active substances are simultaneously extracted, and the utilization rate and economic value of the seaweeds such as the weak plumes are improved.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria comprises the following specific steps:
A. extraction and purification of phycobiliprotein
(1) Mixing the fine and weak plumes according to a feed liquid ratio of 1:1 to 1:1.5 Adding deionized water into the mixture for breaking wall and crushing (the mass volume ratio g is mL), putting the mixture into a refrigerator at the temperature of minus 20 ℃ to minus 30 ℃, repeatedly freezing and thawing for 3 to 5 times, filtering the mixture by using a 50-200 mesh (preferably 100 mesh) fine screen to remove algae residues, and obtaining a phycobiliprotein crude extract from the filtrate;
(2) Adding ammonium sulfate with the volume of 60-100% of the phycobiliprotein crude extract into the phycobiliprotein crude extract for salting out;
(3) Centrifuging the salting-out solution in the step (2) for 5-10 min at the rotation speed of 4000-6000 rpm and the temperature of 2-8 ℃, removing supernatant, collecting precipitate, adding 100-300 mL of deionized water for dissolution, putting the obtained solution into a dialysis bag for dialysis, changing water every 2-4 h until no sulfate radical exists in the dialyzate;
(4) Loading the dialyzate of the step (3) to a phenyl-agarose swelling column at a speed of 1-3 mL/min, eluting with 0.5M ammonium sulfate until the dialyzate is colorless, cleaning the adsorbed impurity proteins and the non-adsorbed phycoerythrin on the swelling column, eluting with 0.2-0.025M ammonium sulfate gradient, and collecting the eluent;
(5) Dialyzing the eluent to remove salt, concentrating until the volume of the dextran G-150 gel chromatographic column is 1% -5%, loading, eluting with 0.05M phosphate buffer, and collecting phycoerythrin eluent; obtaining purified phycoerythrin;
(6) Carrying out ultraviolet absorption spectrum, fluorescence spectrum, SDS-PAGE and Native-PAGE measurement on the phycoerythrin obtained by purification;
B. carrageenan is extracted from algae residue for extracting phycobiliprotein
(1) Putting the algae residue after extracting phycobiliprotein into a beaker, and mixing the algae residue (wet weight) with potassium hydroxide according to a feed liquid ratio of 1:1 to 1:5 (mass volume ratio g: mL) adding alkali liquor with mass concentration of 4% -8%, and water-bathing for 1-3 h at 50-80 ℃;
(2) Filtering with 50-200 mesh (preferably 100 mesh) bolting silk, pickling algae residue with 0.5% acetic acid to pH 7-8, bleaching algae residue with 0.5% sodium hypochlorite, and washing algae residue with deionized water to neutrality;
(3) And then mixing the algae residue with deionized water according to a feed liquid ratio of 1:1 to 1:5 (mass volume ratio g: mL), 70-100 ℃, water bath for 2-4 h;
(4) Filtering the extracting solution (3) while the extracting solution is hot, freezing for 24-72 h after solidification at normal temperature, melting and dehydrating, and drying at 50-70 ℃ to obtain carrageenan;
(5) And (5) measuring the yield, the sulfate group and the infrared spectrum of the prepared carrageenan.
The following describes the invention in part:
the pH of the phosphate buffer=7.
The glucan G-150 gel is soaked in deionized water for more than 24 hours and fully swells.
Compared with the prior art, the invention has the beneficial technical effects that:
1. expands the sources of extraction raw materials of phycoerythrin and carrageenan, and enriches the research of active substances in the fine and weak plumes.
2. The existing phycoerythrin has higher separation and purification cost, and has no mass production in China, the method has the advantages of obtaining the phycoerythrin with the reagent grade purity of 4.19 by twice separation and purification through chromatographic columns, having the yield of 0.06mg/g, high recovery rate, being suitable for large-scale extraction and purification of the phycoerythrin, and being simple and convenient to operate.
3. The extraction of phycobiliprotein residues and carrageenan is carried out, the yield is 19.60% (dry weight), the operation is simple, the comprehensive utilization is realized, two active substances are extracted at the same time, and the utilization rate and the economic value of the fine and weak red feather are improved.
Drawings
FIG. 1 shows the absorption spectrum of the crude extract of fine and weak Solieria pinnatifida of example 1 at 250-700 nm.
FIG. 2 shows the absorption spectrum and fluorescence spectrum of reagent grade phycoerythrin purified from fine and weak Solieria in example 1 at 250-700 nm.
FIG. 3 shows SDS-PAGE and Native-PAGE of the purified fine and weak Solieria pinnatifida of example 1 to obtain phycoerythrins.
FIG. 4 is a FTIR spectrum of carrageenan extracted from the residue of the fine and weak Solieria triangularis of example 1.
Detailed Description
For a better understanding of the essence of the present invention, the following examples of the present invention will be used to illustrate a method for the comprehensive extraction of phycobiliprotein and carrageenan from weak peaked vegetable, but the present invention is not limited to this example.
Example 1: method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumes
The preparation method comprises the following steps:
A. extraction and purification of phycobiliprotein
(1) Weighing 500g of fresh fine and weak plumes with wet weight, cleaning, putting into a wall breaking machine for crushing, adding 600mL of deionized water, putting into a refrigerator at the temperature of minus 20 ℃ for repeated freezing and thawing for 3 times, filtering with 100-mesh fine screening silk to remove algae residues, and obtaining a crude phycobiliprotein extract, wherein the algae residues are reserved in the refrigerator at the temperature of minus 20 ℃.
(2) Adding 70% ammonium sulfate into phycobiliprotein crude extract to carry out salting-out.
(3) Centrifuging the salting-out solution in the step (2) at a rotation speed of 5000rpm and a temperature of 4 ℃ for 6min, removing supernatant, collecting phycobiliprotein precipitate, adding certain amount of water for dissolution, putting the solution into a dialysis bag for dialysis, changing water every 2h until barium chloride is added and no precipitate indicates that no sulfate ion exists, obtaining 350mL of crude extract, and calculating 112mg of phycoerythrin and 0.35 purity according to a formula.
(4) Loading the dialyzate of the step (3) to a phenyl-agarose swelling column at the speed of 2.5mL/min, eluting with 0.5M ammonium sulfate until the dialyzate is colorless, cleaning the heteroprotein adsorbed on the swelling column and the non-adsorbed phycoerythrin, eluting the phycoerythrin with 0.2M, 0.15M, 0.1M, 0.05M and 0.025M ammonium sulfate gradient, collecting the eluent, and calculating to obtain 25.09mg of phycoerythrin in the eluent, wherein the yield is 0.07mg/g, the purity is 2.77, and the recovery rate is 30.43%.
(5) Dialyzing the eluent to remove salt, ultrafiltering and concentrating to 13mL, loading to a dextran G-150 gel chromatographic column, eluting with 0.05M phosphoric acid buffer solution, collecting phycoerythrin eluent, and calculating to obtain 20.7mg of phycoerythrin in the eluent, with a yield of 0.06mg/G, a purity of 4.19 and a recovery rate of 26.09%.
(6) The phycoerythrin obtained by purification is measured by ultraviolet absorption spectrum, fluorescence spectrum, SDS-PAGE and Native-PAGE.
B. Carrageenan is extracted from algae residue for extracting phycobiliprotein
(1) The ratio of the fine and weak red feather herb residues after extracting phycoerythrin is 1:2 is added with 6.5 percent of potassium hydroxide solution and is treated by water bath alkali at 62 ℃ for 2 hours.
(2) Filtering (1), pickling the fine and weak solanum torvum residue with 0.5% acetic acid to pH 7-8, bleaching the residue with 0.5% sodium hypochlorite for 5min, washing the residue with deionized water to neutrality, filtering the residue, and then, according to a feed liquid ratio of 1:2 adding deionized water, and water-bathing at 90 ℃ for 3 hours.
(3) Filtering the solution (2) by using bolting silk, and collecting filtrate.
(4) Freezing overnight after the filtrate is solidified at normal temperature, melting and dehydrating, and then drying at 60 ℃ to obtain the carrageenan. The weighing yield was 19.60% (dry weight).
(5) And (5) carrying out sulfate radical content and FTIR characterization measurement on the dried carrageenan. The sulfate content was 7.89%. As can be seen from the FTIR spectra, the characteristic peaks of carrageenan were extracted: the absorption peak of CH is 2920cm -1 Is the stretching vibration on the six-membered ring, and the absorption peak of OH is 3400cm -1 Nearby, the absorption peak of 3, 6-galactoside group is 930cm -1 At the place, the characteristic peak of the COC group is 1100cm -1 Total SO 4 The absorption peak of (C) is 1214cm -1 At 847cm -1 Has characteristic absorption peak, which indicates that the C4 of the (1, 3) -beta-D-galactose of carrageenan has SO 4 Indicating that the main component is k-carrageenan, at 803cm -1 Has obvious characteristic peak, which indicates that the C2 of the 3, 6-internal ether-D-galactose has SO 4 Iota-carrageenan is present. Comparative example 1:
the preparation method comprises the following steps:
A. extraction and purification of phycobiliprotein
(1) Weighing 500g of fresh fine and weak plumes with wet weight, cleaning, crushing in a wall breaking machine, adding 650mL of deionized water, repeatedly freezing and thawing for 3 times in a refrigerator at-20 ℃, filtering with fine screening silk to remove algae residues, and obtaining crude extract of phycobiliprotein, wherein the algae residues are reserved in the refrigerator at-20 ℃.
(2) Adding 70% ammonium sulfate into phycobiliprotein crude extract to carry out salting-out.
(3) Centrifuging the salting-out solution in the step (2) at a rotation speed of 5000rpm and a temperature of 4 ℃ for 6min, removing supernatant, collecting phycobiliprotein precipitate, adding certain amount of water for dissolution, putting the solution into a dialysis bag for dialysis, changing water every 2h until barium chloride is added and no precipitate is generated, thus indicating that no sulfate ions exist. 300mL of crude extract is obtained, 115mg of phycoerythrin is contained and the purity is 0.35 according to the formula.
(4) Loading the dialyzate of the step (3) to a phenyl-agarose swelling column at a speed of 2.5mL/min, eluting part of phycoerythrin without being combined with the column at a speed of too high, eluting the phycoerythrin to be colorless by using 0.5M ammonium sulfate at a speed of 2.5mL/min, eluting the phycoerythrin by using a gradient of 0.1-0.0025M ammonium sulfate, mixing the hetero protein with the phycoerythrin, eluting, collecting the eluent according to the column volume, measuring the absorption spectrum after dialysis, and calculating to obtain the total phycoerythrin content of 23.74mg in the eluent, wherein the yield is 0.05mg/g, the purity is 2.43 and the recovery rate is 21.74%. The sample is loaded and eluted at a high speed, the purification effect is greatly reduced, the purity is reduced, and the phycoerythrin loss rate is high.
(5) Dialyzing the eluent to remove salt, ultrafiltering and concentrating to 5% of column volume, loading onto dextran G-150 gel chromatographic column, eluting with 0.05M phosphoric acid buffer solution, collecting phycoerythrin eluent, dialyzing, measuring absorption spectrum, and calculating to obtain phycoerythrin 17.50mg in eluent with yield of 0.035mg/G, purity of 3.62 and recovery rate of 14.00%.
B. Carrageenan is extracted from algae residue for extracting phycobiliprotein
((1) adding 6.5% potassium hydroxide solution into the fine and weak solanum torvum residue after extracting phycoerythrin according to the feed-liquid ratio of 1:2, and carrying out alkali treatment in a water bath at 90 ℃ for 2h.
(2) Filtering (1), pickling the fine and weak solanum torvum residue with 0.5% acetic acid to pH 7-8, bleaching the residue with 0.5% sodium hypochlorite for 5min, washing the residue with deionized water to neutrality, filtering the residue, and then, according to a feed liquid ratio of 1:2 adding deionized water, and water-bathing at 90 ℃ for 3 hours.
(3) Filtering the solution (2) by using bolting silk, and collecting filtrate. (4) Freezing overnight after the filtrate is solidified at normal temperature, melting and dehydrating, and then drying at 60 ℃ to obtain the carrageenan. The weighing yield was 10.04% (dry weight), and too high a temperature of alkali extraction would degrade the carrageenan during alkali treatment, resulting in reduced yields.
Comparative example 2:
the preparation method comprises the following steps:
A. extraction and purification of phycobiliprotein
(1) Weighing 500g of fresh fine and weak plumes with wet weight, cleaning, putting into a wall breaking machine for crushing, adding 600mL of deionized water, putting into a refrigerator at the temperature of minus 20 ℃ for repeated freezing and thawing for 3 times, filtering with 100-mesh fine screening silk to remove algae residues, and obtaining a crude phycobiliprotein extract, wherein the algae residues are reserved in the refrigerator at the temperature of minus 20 ℃.
(2) Adding 70% ammonium sulfate into phycobiliprotein crude extract to carry out salting-out.
(3) Centrifuging the salting-out solution in the step (2) at a rotation speed of 5000rpm and a temperature of 4 ℃ for 6min, removing supernatant, collecting phycobiliprotein precipitate, adding certain amount of water for dissolution, putting the solution into a dialysis bag for dialysis, changing water every 2h until barium chloride is added and no precipitate indicates that no sulfate ion exists, obtaining 350mL of crude extract, and calculating 112mg of phycoerythrin and 0.35 purity according to a formula.
(4) Loading the dialyzate of the step (3) to a phenyl-agarose swelling column at the speed of 2.5mL/min, eluting with 0.5M ammonium sulfate until the dialyzate is colorless, cleaning the heteroprotein adsorbed on the swelling column and the non-adsorbed phycoerythrin, eluting the phycoerythrin with 0.2M, 0.15M, 0.1M, 0.05M and 0.025M ammonium sulfate gradient, collecting the eluent, and calculating to obtain 25.09mg of phycoerythrin in the eluent, wherein the yield is 0.07mg/g, the purity is 2.77, and the recovery rate is 30.43%.
(5) Dialyzing the eluent to remove salt, loading the eluent to a Q-anion chromatographic column, performing gradient elution by using 0.05M PBS buffer solution (containing 0.05M, 0.1M, 0.15M and 0.2M sodium chloride) with pH of 7, collecting phycoerythrin eluent, measuring absorption spectrum, and calculating to obtain 21.13mg of phycoerythrin in the eluent, wherein the yield is 0.04mg/g, the purity is 3.85, and the recovery rate is 17.39%.
B. Carrageenan is extracted from algae residue for extracting phycobiliprotein
(1) The ratio of the fine and weak red feather herb residues after extracting phycoerythrin is 1:2, adding 6.5% potassium hydroxide solution, and performing alkali treatment in a water bath at 60 ℃ for 6 hours.
(2) Bleaching the algae residue for 5min by using 0.5% sodium hypochlorite, washing the algae residue to be neutral by using deionized water, filtering the algae residue, and then, according to a feed-liquid ratio of 1:2 adding deionized water, and water-bathing at 90 ℃ for 3 hours.
(3) Filtering the solution (2) by using bolting silk, and collecting filtrate.
(4) Freezing overnight after the filtrate is solidified at normal temperature, melting and dehydrating, and then drying at 60 ℃ to obtain the carrageenan. The weighing yield was 8.00% (dry weight) and the gum was obtained to contain a part of pigment material. Long-term alkaline extraction can degrade carrageenan during alkaline treatment, resulting in reduced yields.
Claims (4)
1. A method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria is characterized in that:
the method comprises the following specific steps:
A. extracting and purifying phycobiliprotein:
(1) The fine and weak plumes are mixed according to the mass-volume ratio of 1: 1-1: 1.5 adding deionized water, breaking the wall, crushing, putting into a refrigerator at-20 to-30 ℃, repeatedly freezing and thawing for 3-5 times, filtering with 50-200 meshes to remove algae residues, and obtaining a phycobiliprotein crude extract from the filtrate;
(2) Adding ammonium sulfate accounting for 60% -100% of the volume of the phycobiliprotein crude extract into the phycobiliprotein crude extract for salting out;
(3) Centrifuging the salting-out solution obtained in the step (2) for 5-10 min at the rotation speed of 4000-6000 rpm and the temperature of 2-8 ℃, removing supernatant, collecting precipitate, adding 100-300 mL of deionized water for dissolution, putting the obtained solution into a dialysis bag for dialysis, changing water every 2-4 h until no sulfate radical exists in the dialyzate;
(4) Loading the dialysate obtained in the step (3) to a phenyl-agarose swelling column at a speed of 1-3 mL/min, eluting with 0.5-M ammonium sulfate until the dialysate is colorless, cleaning the adsorbed impurity proteins and the unadsorbed phycoerythrin on the swelling column, eluting with 0.2-0.025M ammonium sulfate gradient, and collecting the eluent;
(5) Dialyzing the eluent to remove salt, concentrating to 1% -5% of the volume of the dextran G-150 gel chromatographic column, loading, eluting with 0.05M phosphate buffer, and collecting phycoerythrin eluent; obtaining purified phycoerythrin;
(6) Carrying out ultraviolet absorption spectrum, fluorescence spectrum, SDS-PAGE and Native-PAGE measurement on the phycoerythrin obtained by purification;
B. extracting carrageenan from the phycobiliprotein extracted algae residues:
(1) Putting the algae residues after the extraction of phycobiliprotein into a container, wherein the mass volume ratio of the wet weight material liquid of the algae residues is 1: 1-1: 5, adding potassium hydroxide alkali liquor with the mass concentration of 4% -8%, and carrying out water bath for 1-3 hours at 50-80 ℃;
(2) Filtering with 50-200 mesh bolting silk, pickling algae residue with acetic acid with concentration of 0.5% to pH 7-8, bleaching algae residue with sodium hypochlorite with concentration of 0.5%, and washing algae residue with deionized water to neutrality;
(3) And then the algae residue and deionized water are mixed according to the mass volume ratio of 1: 1-1: 5 g: mL; water bath is carried out for 2-4 hours at 70-100 ℃;
(4) Filtering the extracting solution obtained in the step (3) while the extracting solution is hot, freezing for 24-72 hours after solidification at normal temperature, melting and dehydrating, and drying at 50-70 ℃ to obtain carrageenan;
(5) And (5) measuring the yield, the sulfate group and the infrared spectrum of the prepared carrageenan.
2. The method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria as claimed in claim 1, wherein the method comprises the following steps: the glucan G-150 gel is soaked in deionized water for more than 24 and h, and is fully swelled.
3. The method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria as claimed in claim 1, wherein the method comprises the following steps: the pH of the phosphate buffer=7.
4. The method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria as claimed in claim 1, wherein the method comprises the following steps: and (3) filtering in the step (1) and removing algae residues by using 100-mesh fine screening silk.
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