CN105111278A - Method for preparing crude phycobiliprotein and crude polysaccharide from porphyra yezoensis - Google Patents

Abstract

The invention provides a method for preparing crude phycobiliprotein and crude polysaccharide from porphyra yezoensis. The novel method for preparing crude phycobiliprotein and crude polysaccharide simultaneously comprises steps as follows: fresh and wet porphyra is taken as a raw material, harvested porphyra yezoensis is washed, drained and subjected to centrifugal dewatering processing, and mixing swelling, wet-method ultrafine grinding performed by a colloid mill combined homogenizer, salting-out and other procedures are performed. According to the method, drying processing of the fresh and wet porphyra in the preparation process of phycobiliprotein is eliminated, a prepared crude phycobiliprotein product contains 34.3%-38.9% of phycobiliprotein on the dry basis, and the yield of phycobiliprotein ranges from 4.59% to 4.71%. The method is suitable for industrial application, the crude phycobiliprotein product prepared from porphyra can be taken as a preparation raw material of high-purity porphyra phycobiliprotein, and the prepared crude polysaccharide can be taken as a preparation raw material of polysaccharide.

Description

A method for preparing crude phycobiliprotein and crude polysaccharide from Porphyra zebra

technical field

The invention relates to a method for preparing phycobiliprotein and polysaccharide, in particular to a method for preparing crude phycobiliprotein and crude polysaccharide by using Porphyra zebra.

Background technique

Porphyra is a general term for macroalgae of Rhodophyta ( Rhodophyta ) and Porphyra ( Porphyra ), and it is one of the most important economical algae. Porphyrayezoensis and Porphyrahaitanensis are two major species of laver in China. Seaweed is high in protein, low in fat, rich in mineral elements and multivitamins, and is considered by Chinese medicine as one of the precious natural seaweed health foods. At present, laver processing products on the market are mainly dried laver cakes, instant seaweed and brewed laver soup, resulting in the status quo of single products, low added value and slow updating and upgrading, which restricts the sustainable development and growth of the laver industry.

Dried seaweed contains 25-50% protein and 20-40% sugar. Phycobiliproteins and polysaccharides are important components of laver. Phycobiliprotein is a water-soluble, naturally active pigment protein, mainly composed of phycoerythrin, phycocyanin and allophycocyanin. Oxidation, anti-aging and other biological activities, can be used as a photosensitizer for tumor treatment, and can be used as an immunofluorescence marker for special molecular positioning, detection and medical diagnosis. Phycobiliprotein has a broad potential market in the fields of food, medicine and cosmetics. At present, the price of phycobiliprotein products invested and developed by foreign companies can reach more than $100/mg. Porphyra polysaccharides have biological functions such as immune regulation, lowering blood fat, lowering blood sugar, anticoagulation, anti-inflammation, anti-fatigue, anti-aging, anti-radiation, anti-influenza virus, anti-cancer, etc. It also has broad applications in the fields of food, medicine and cosmetics prospect. However, the current development of Porphyra phycobiliproteins and polysaccharides is generally done with dry Porphyra as raw material, or after drying fresh and wet Porphyra for follow-up treatment. However, only 1 kg of dry laver can be obtained after drying 10-13 kg of fresh and wet laver, and the drying process will consume a lot of heat energy and produce leftovers, and will also cause protein denaturation and affect the extraction and activity of phycobiliprotein. Therefore, it is of great significance and industrialization prospect to develop new technologies for the preparation of high value-added phycobiliproteins and polysaccharides directly using fresh and wet laver as raw materials.

Contents of the invention

The technical problem to be solved by the present invention is to provide a new method for preparing crude phycobiliprotein and crude polysaccharide from Porphyra zebra with reasonable method design and strong operability in view of the deficiencies in the existing Porphyra phycobiliprotein and polysaccharide preparation technology. method.

The technical problem to be solved by the present invention is achieved through the following technical solutions. The present invention is a method for preparing crude phycobiliproteins and crude polysaccharides from Porphyra variegata, which is characterized in that the main steps of the method include:

(1) Wash, drain, and centrifugally dehydrate the harvested Porphyra variegata to obtain fresh and wet laver with a water content of 79.5-81.2%; add potassium dihydrogen phosphate-sodium hydroxide with a pH of 8.0 to the fresh and wet laver buffer solution, the mass ratio of buffer solution and seaweed is 0.8~1:1; homogenate to a slurry state, and then swell the slurry material at 38~42°C for 8~10 hours to obtain the first material.

(2) Treat the first material with a colloid mill, then dilute the treated material with an appropriate amount of water and then process it with a high-pressure homogenizer at 75-100Mpa to obtain the second material; centrifuge the second material to obtain The first supernatant and the first precipitate; add ammonium sulfate to the first supernatant, then let stand at 4-10°C for 10-12 hours, and then centrifuge to obtain the second supernatant and the second The precipitate; the second precipitate is the crude phycobiliprotein product; the first precipitate and the second supernatant are the crude polysaccharide.

The method for preparing crude phycobiliprotein and crude polysaccharide by using Porphyra variegata according to the present invention, the further preferred technical scheme is as follows:

1. In step (1): when the dehydrated fresh and wet laver cannot be homogenized in time, place it in an environment with a temperature ≤ -18°C for freezing and preservation, and then add buffer for homogenization.

2. In step (1): washing, draining, and centrifugal dehydration of the harvested Porphyra variegata to obtain fresh wet Porphyra with a water content of 80-81%.

3. In step (1): the mass ratio of buffer solution to seaweed is 0.9:1; homogenate to a slurry state, and then swell the slurry material at 40° C. for 9 hours to obtain the first material.

4. In step (2): when the material is diluted with water, the mass ratio of water to the first material is 0.8~1:3.

5. In step (2): when ammonium sulfate is added to the first supernatant, the saturation of ammonium sulfate gradually reaches 45-60%.

6. In step (2): the material treated by the colloid mill is diluted with an appropriate amount of water and then processed by a high-pressure homogenizer at 85-90Mpa.

7. In step (2): adding ammonium sulfate to the first supernatant, then standing at 4-6°C for 10 hours, and then performing centrifugation to obtain the second supernatant and the second precipitate.

Among the present invention: the dry matter content analysis can adopt 105 ℃ constant weight method; The mensuration of phycoerythrin, phycocyanin, allophycocyanin content in crude phycobiliprotein product adopts Gao Hongfeng (Gao Hongfeng. In different growth stages of Porphyra laver Changes in the content of phycobiliproteins [J]. Ocean and Limnology, 1993:645-648.) method: use the Lowry method to determine the concentration of pure phycobiliproteins, and then determine the maximum concentration of mixed phycobiliproteins in each phycobiliprotein component The absorbance at the absorption wavelength is calculated from the extinction coefficient of each phycobiliprotein according to the Lambert-Beer law to calculate the content of each phycobiliprotein in the mixed phycobiliprotein solution.

In the method of the present invention: the purpose of cleaning, draining, and centrifugal dehydration of the harvested Porphyra variegata is to remove impurities in the Porphyra and reduce the water content of Porphyra, which is conducive to reducing the swelling liquid (potassium dihydrogen phosphate-sodium hydroxide buffer solution), and the effect of improving homogenization and swelling. The purpose of subjecting the first material to colloid mill treatment is to preliminarily break the cell wall of laver to dissolve the protein (colloid mill treatment can make the finished product particle size reach 2-50 μm), and at the same time make the material use subsequent high-pressure homogenization treatment. The purpose of diluting the material treated by the colloid mill with water is to adjust the fluid properties of the material to facilitate subsequent high-pressure homogeneous treatment. The purpose of the 75-100Mpa high-pressure homogenizer treatment is to deeply break the cell wall of laver to fully dissolve the protein (the homogenizer treatment can make the finished product particle size reach 0.01-2μm), so as to achieve the effect of increasing the protein yield.

Compared with prior art, the beneficial effect of the inventive method is as follows:

The present invention uses fresh and wet laver as raw material and adopts modern food development technology. First, the laver is swelled to change the tissue structure of the laver, so as to facilitate subsequent wet ultrafine pulverization (colloid mill combined with homogenizer treatment) to dissolve protein; and then , on the basis of the swelling treatment, a colloid mill and a high-pressure homogenizer are combined for wet ultrafine pulverization treatment, thereby destroying the laver cell wall and fully dissolving the laver protein, so as to utilize the subsequent preparation of phycobiliprotein. The present invention uses fresh and wet laver as raw material for the first time, establishes the preparation technology of laver phycobiliprotein and polysaccharide based on swelling and wet superfine pulverization (colloid mill combined with homogenizer treatment), and clarifies the process and parameters of the whole technology . At present, for the development of laver phycobiliproteins and polysaccharides, the common practice is to use dried laver as raw material or to dry fresh and wet laver for follow-up treatment, which leads to only 1 kg of dry laver after drying 10-13 kg of fresh and wet laver. Porphyra, and the drying process will consume a lot of heat energy and produce leftovers, and will also cause protein denaturation and affect the extraction and activity of phycobiliproteins. Therefore, the present invention avoids this shortcoming, and provides a method for preparing New approach to phycobiliproteins and polysaccharides. The crude phycobiliprotein product prepared by this method contains 16.4-18.9% of dry matter, 34.3-38.9% of phycobiliprotein (phycoerythrin + phycocyanin + allophycocyanin) (calculated on a dry basis), and the phycobiliprotein The yield (the mass percentage of phycobiliprotein contained in the prepared crude phycobiliprotein product in the consumed dehydrated laver) is 4.59-4.71%. The invention uses fresh and wet laver as raw material, integrates swelling, wet superfine pulverization (colloid mill combined with homogenizer treatment), salting out and other processes, and constructs a new method for preparing laver crude phycobiliprotein and crude polysaccharide. Both the colloid mill and the homogenizer are commonly used equipment in the food industry at present, so the present invention is suitable for industrial application. The prepared first precipitate and the second supernatant are crude polysaccharides, which can be further prepared into laver polysaccharides; the prepared crude laver phycobiliprotein products can be used as raw materials for preparing high-purity laver phycobiliproteins.

Detailed ways

The specific technical solutions of the present invention are further described below, so that those skilled in the art can further understand the present invention.

Embodiment 1, a method for preparing crude phycobiliprotein and crude polysaccharide by using Porphyra zebra, the main steps of the method include:

(1) Wash, drain, and centrifugally dehydrate the harvested Porphyra variegata to obtain fresh wet laver with a water content of 79.5%; add potassium dihydrogen phosphate-sodium hydroxide buffer solution with a pH of 8.0 to the fresh wet laver , the mass ratio of the buffer solution to the seaweed is 0.8:1; homogenize to a slurry state, and then swell the slurry material at 38° C. for 8 hours to obtain the first material.

(2) Treat the first material with a colloid mill, then dilute the treated material with an appropriate amount of water and then process it with a high-pressure homogenizer at 75Mpa to obtain the second material; centrifuge the second material to obtain the first supernatant and the first precipitate; add ammonium sulfate to the first supernatant, then stand at 4°C for 10 hours, then centrifuge to obtain the second supernatant and the second precipitate; the The second precipitate is the crude phycobiliprotein product; the first precipitate and the second supernatant are crude polysaccharides.

In step (2) of this embodiment: when the material is diluted with water, the mass ratio of water to the first material is 0.8:3; when ammonium sulfate is added to the first supernatant, the saturation of ammonium sulfate gradually reaches 45%.

Embodiment 2, a method for preparing crude phycobiliprotein and crude polysaccharide by using Porphyra zebra, the main steps of the method include:

(1) Wash, drain, and centrifugally dehydrate the harvested Porphyra variegata to obtain fresh wet laver with a water content of 81.2%; add potassium dihydrogen phosphate-sodium hydroxide buffer solution with a pH of 8.0 to the fresh wet laver , the mass ratio of the buffer solution to the seaweed is 1:1; homogenize to a slurry state, and then swell the slurry material at 42° C. for 10 hours to obtain the first material.

(2) Treat the first material with a colloid mill, then dilute the treated material with an appropriate amount of water and then process it with a high-pressure homogenizer at 100Mpa to obtain the second material; centrifuge the second material to obtain the first supernatant and the first precipitate; adding ammonium sulfate to the first supernatant, then standing at 10°C for 12 hours, and then centrifuging to obtain the second supernatant and the second precipitate; the The second precipitate is the crude phycobiliprotein product; the first precipitate and the second supernatant are crude polysaccharides.

In step (2) of this embodiment: when the material is diluted with water, the mass ratio of water to the first material is 1:3; when ammonium sulfate is added to the first supernatant, the saturation of ammonium sulfate gradually reaches 60%.

Embodiment 3, a method for preparing crude phycobiliprotein and crude polysaccharide by using Porphyra zebra, the main steps of the method include:

(1) Wash, drain, and centrifugally dehydrate the harvested Porphyra variegata to obtain fresh wet laver with a water content of 80%; add potassium dihydrogen phosphate-sodium hydroxide buffer solution with a pH of 8.0 to the fresh wet laver , the mass ratio of the buffer solution to the seaweed is 0.9:1; homogenize to a slurry state, and then swell the slurry material at 40° C. for 9 hours to obtain the first material.

(2) Treat the first material with a colloid mill, then dilute the treated material with an appropriate amount of water and then process it with a high-pressure homogenizer at 85Mpa to obtain the second material; centrifuge the second material to obtain the first supernatant and the first precipitate; adding ammonium sulfate to the first supernatant, then standing at 6°C for 11 hours, and then centrifuging to obtain the second supernatant and the second precipitate; the The second precipitate is the crude phycobiliprotein product; the first precipitate and the second supernatant are crude polysaccharides.

In step (2) of this embodiment: when the material is diluted with water, the mass ratio of water to the first material is 0.9:3; when ammonium sulfate is added to the first supernatant, the saturation of ammonium sulfate gradually reaches 50%.

Embodiment 4, a method for preparing phycobiliproteins and polysaccharides utilizing fresh and wet Porphyra variegata, the main steps of which include:

(1) Wash, drain, and centrifugally dehydrate the harvested Porphyra variegata to obtain fresh wet laver with a water content of 80.3%; add potassium dihydrogen phosphate of pH 8.0 to the dehydrated fresh wet laver – hydroxide Sodium buffer solution, the mass ratio of the buffer solution and laver is 1:1; homogenize the mixture to a slurry state, and then swell the slurry material at 40° C. for 9 hours to obtain the first material.

(2) Treat the first material with a colloid mill, then dilute the material after the colloid mill treatment with water, so that the mass ratio of water and the first material is 1:3, and then process it with a high-pressure homogenizer at 80Mpa to obtain the first The second material; the second material is centrifuged to obtain the first supernatant and the first precipitate; ammonium sulfate is added to the first supernatant so that the saturation of ammonium sulfate gradually reaches 45%, and then static at 4°C Set for 12 hours, then centrifuge to obtain the second supernatant and the second precipitate; the second precipitate is the crude phycobiliprotein product; the first precipitate and the second supernatant are For crude polysaccharides.

In this embodiment, when the dehydrated fresh wet laver cannot be homogenized in time, it can be frozen and preserved at a temperature ≤ -18° C., and then added with a buffer for homogenization.

Embodiment 5, a method for preparing phycobiliproteins and polysaccharides utilizing fresh and wet Porphyra variegata, the main steps of which include:

(1) Wash, drain, and centrifugally dehydrate the harvested Porphyra variegata to obtain fresh wet laver with a water content of 80.8%; add potassium dihydrogen phosphate of pH 8.0 to the dehydrated fresh wet laver – hydroxide Sodium buffer solution, the mass ratio of the buffer solution and laver is 0.9:1; homogenize the mixture to a slurry state, and then swell the slurry material at 39° C. for 10 hours to obtain the first material.

(2) Treat the first material with a colloid mill, then dilute the material after the colloid mill treatment with water, so that the mass ratio of water and the first material is 0.9:3, and then process it with a high-pressure homogenizer at 90Mpa to obtain the first The second material; the second material is centrifuged to obtain the first supernatant and the first precipitate; ammonium sulfate is added to the first supernatant so that the saturation of ammonium sulfate gradually reaches 60%, and then static at 6°C Leave it for 10 hours, then carry out centrifugation to obtain the second supernatant and the second precipitate; the second precipitate is the crude phycobiliprotein product; the first precipitate and the second supernatant are For crude polysaccharides.

In this embodiment, when the dehydrated fresh wet laver cannot be homogenized in time, it can be frozen and preserved at a temperature ≤ -18° C., and then added with a buffer for homogenization.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention to other forms. Any skilled person who is familiar with this profession may use the technical content disclosed above to change or modify the equivalent of equivalent changes. Example. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims (8)

1. a method utilizing Porphyra zebra to prepare crude phycobiliprotein and crude polysaccharide, is characterized in that, the main steps of the method comprise: (1) Wash, drain, and centrifugally dehydrate the harvested Porphyra variegata to obtain fresh and wet laver with a water content of 79.5-81.2%; add potassium dihydrogen phosphate-sodium hydroxide with a pH of 8.0 to the fresh and wet laver The buffer solution, the mass ratio of buffer solution and seaweed is 0.8~1:1; homogenate to a slurry state, and then swell the slurry material at 38~42°C for 8~10 hours to obtain the first material; (2) Treat the first material with a colloid mill, then dilute the treated material with an appropriate amount of water and then process it with a high-pressure homogenizer at 75-100Mpa to obtain the second material; centrifuge the second material to obtain The first supernatant and the first precipitate; add ammonium sulfate to the first supernatant, then let stand at 4-10°C for 10-12 hours, and then centrifuge to obtain the second supernatant and the second The precipitate; the second precipitate is the crude phycobiliprotein product; the first precipitate and the second supernatant are the crude polysaccharide. 2. The method for preparing crude phycobiliproteins and crude polysaccharides from Porphyra zebra according to claim 1, characterized in that in step (1): when the dehydrated fresh and wet Porphyra cannot be homogenized in time, the After it is placed in an environment with a temperature ≤ -18°C, it is frozen and kept fresh, and then buffer solution is added for homogenization. 3. The method for preparing crude phycobiliprotein and crude polysaccharide by using Porphyra zebra according to claim 1, characterized in that in step (1): washing, draining, and centrifugal dehydration of the harvested Porphyra zebra, Fresh wet laver with a water content of 80-81% is obtained. 4. The method for preparing crude phycobiliprotein and crude polysaccharide from Porphyra zebra according to claim 1, characterized in that, in step (1): the mass ratio of buffer solution to Porphyra is 0.9:1; homogenate to slurry shape, and then the slurry material was swollen at 40° C. for 9 hours to obtain the first material. 5. The method for preparing crude phycobiliprotein and crude polysaccharide from Porphyra zebra according to claim 1, characterized in that, in step (2): when the material is diluted with water, the mass ratio of water to the first material is 0.8~ 1:3. 6. The method for preparing crude phycobiliprotein and crude polysaccharide from Porphyra zebra according to claim 1, characterized in that, in step (2): when ammonium sulfate is added to the first supernatant, the ammonium sulfate is saturated Gradually reach 45~60%. 7. The method for preparing crude phycobiliprotein and crude polysaccharide by using Porphyra zebra according to claim 1 or 5, characterized in that, in step (2): the material after colloid mill treatment is diluted with an appropriate amount of water at 85 ~90Mpa for high-pressure homogenizer treatment. 8. The method for preparing crude phycobiliprotein and crude polysaccharide from Porphyra zebra according to claim 1 or 6, characterized in that, in step (2): adding ammonium sulfate to the first supernatant, and then Stand at ~6°C for 10 hours, and then perform centrifugation to obtain a second supernatant and a second precipitate.