WO2023115326A1 - Composition for enhancing photothermal stability of phycobiliproteins, and preparation method therefor and use thereof - Google Patents

Abstract

A composition for enhancing the photothermal stability of phycobiliproteins, comprising an extract of blue-green algae and an extract of red algae. The present invention further relates to a method for preparing the composition for enhancing the photothermal stability of phycobiliproteins. The method comprises: taking blue-green algae biomass, obtaining an extracting solution by means of enzymolysis combined with a hyperosmotic condition, and purifying and drying the extracting solution to obtain an extract powder of blue-green algae; taking red algae biomass, obtaining an extracting solution by means of hot water extraction, and purifying and drying the extracting solution to obtain an extract powder of red algae; and mixing the extract powder of blue-green algae and the extract powder of red algae to obtain the composition. Further disclosed is the use of said composition as an additive for improving the thermostability and photostability of phycobiliproteins. The extract of blue-green algae and the extract of red algae can effectively improve the photostability and thermostability of phycobiliproteins, so as to protect the stability of phycobiliproteins in a high-temperature spray drying process, which prevents phycobiliproteins from being destroyed and inactivated.

Description

Composition for enhancing photothermal stability of phycobiliprotein and its preparation method and application technical field

The invention relates to a composition for enhancing the photothermal stability of phycobiliprotein, as well as a preparation method and application of the composition.

Background technique

Phycobiliprotein (phycobiliprotein) is a natural pigment protein complex with various biological activities such as anti-oxidation, anti-tumor, anti-radiation, enhancing immunity, and fluorescence properties. Common phycobiliproteins include phycoerythrin (phycoerythrin) and Phycocyanin, which widely exists in red algae, cyanobacteria, cryptophyta and some dinoflagellates, has important application value in the fields of medicine, nutrition, medical testing, health care products, photodynamic therapy, food, cosmetics, etc. . Phycobiliproteins are an important part of photosynthesis in algae. They mainly exist in the form of polymers on thylakoid membranes, and phycocyanins often exist in the form of trimers (αβ) ₃ or hexamers (αβ) ₆ , while phycoerythrin often exists in the form of hexamer (αβ) ₆ γ, and the molecular weights of α, β and γ subunits are different, respectively 20kD, 24kDa and 34kDa, so different types of phycobiliproteins have different molecular weights. Phycobiliprotein has good water solubility, and its isoelectric point is usually lower than 6. Since phycocyanin and phycoerythrin have different pigment groups, they have different light absorption characteristics, and the maximum absorption of phycoerythrin is at 545nm , The maximum absorption peak of phycocyanin is at 620nm. Phycobiliproteins with a purity greater than 0.7 are food grade, those with a purity greater than 3 are pharmaceutical grades, and those with a purity greater than 4 are reagent grades. Reagent-grade phycobiliproteins are the most expensive, with prices as high as 1,500 yuan/mg. Phycobiliproteins have special fluorescent activity. Using phycobiliprotein as a fluorescent marker and combining with biotin or monoclonal antibody to prepare fluorescent probes has broad application prospects in the field of medical detection.

Phycobiliproteins have poor photostability and thermal stability, and are easily denatured during preparation, storage and processing. High temperatures exceeding 65°C will destroy the structure of phycobiliproteins. High concentrations of metal ions, organic solvents, pH, Light may destroy the structure of phycobiliprotein. In order to maintain the stability of phycobiliprotein, it is usually necessary to add a certain amount of stabilizer during its production and processing. Commonly used stabilizers include trehalose and citric acid. Trehalose is a non-reducing disaccharide composed of two glucose molecules. It exists widely in different biological cells. When the organism is in adversity, it can maintain the stability of intracellular proteins. It is currently widely used in medicine, food, cosmetics and other industries , such as antibody stabilizers, cosmetic moisturizing ingredients, food sweeteners and quality improvers. Citric acid is a small molecule organic acid that is easily soluble in water and has chelation and pH adjustment properties. Although trehalose and citric acid are widely used in the preparation and processing of phycobiliproteins, their effects on phycobiliproteins are limited to maintaining the stability of their chemical structures, but have no significant effect on their photostability and thermal stability. Enhance the effect. And excessive use of trehalose and citric acid in phycobiliprotein will affect product quality. In addition, excessive citric acid and a temperature as high as 180 degrees during the spray-drying process of phycobiliprotein will cause its fluorescence properties to decrease or even disappear.

Contents of the invention

One of the objects of the present invention is to provide a composition for enhancing the photothermal stability of phycobiliprotein.

The present invention achieves the above object through the following technical solutions: the composition for enhancing the photothermal stability of phycobiliproteins includes extracts of blue algae and red algae.

The respective mass fractions of the cyanobacteria extract and the red algae extract are: 10%-90% of the cyanobacteria extract and 10%-90% of the red algae extract.

The cyanobacteria extract is the extract of cyanobacteria biomass, and the cyanobacteria biomass is spirulina, kudzuba or ground fungus, which can be dry algae or wet algae.

The red algae extract is an extract of red algae biomass, and the red algae biomass is centipede algae, dulse palmate and Gracilaria, which can be dry algae or wet algae.

The second object of the present invention is to provide a preparation method of the composition for enhancing the photothermal stability of phycobiliprotein.

The present invention achieves the above object through the following technical scheme: the preparation method of the composition for enhancing the photothermal stability of phycobiliprotein comprises the following steps:

(1) After the cyanobacteria biomass is subjected to hyperosmotic coupling enzymolysis treatment, it is filtered, and the obtained filtrate is an extract, which is purified and dried to obtain a cyanobacteria extract powder;

(2) get red algae biomass and adopt hot water extraction method to extract, filter, gained filtrate is extract, purify, dry to obtain red algae extract powder;

(3) Take blue algae extract powder and red algae extract powder and mix to obtain.

As an embodiment of the present invention, the hypertonic condition of the hypertonic coupling enzymolysis of the step (1) is 5-300g/L sodium chloride, 2-60g/L calcium chloride and 0.05-0.2mol/L phosphoric acid Combination of any two or three of potassium (sodium) salts, enzymolysis treatment conditions are a compound enzyme system of cellulase and pectinase, and hyperosmotic coupled enzymolysis treatment at 4 degrees for 7-9 hours. The mass ratio of cellulase and pectinase in the compound enzyme system of cellulase and pectinase is 1:1.

The purification in the step (1) and step (2) is ultrafiltration purification. As an example, a filter membrane with a pore size of 10 kDa is used for ultrafiltration treatment.

The drying in the step (1) and step (2) can be spray drying or low-temperature freeze-drying of the filtrate.

In the step (2), the red algal biomass is first broken into smaller fragments to facilitate subsequent hot water extraction.

In the step (2), the hot water extraction is heated at 70-90 degrees for 3-5 hours.

The cyanobacteria biomass is spirulina, Gexianmi or ground fungus, and can be dry algae or wet algae.

The red algae biomass is centipede algae, dulse palmate and Gracilaria, which can be dry algae or wet algae.

The third object of the present invention relates to the application of the composition for enhancing the photothermal stability of phycobiliprotein. Specifically, it relates to the application of the composition for enhancing the photothermal stability of phycobiliprotein as an additive for improving the thermal stability and light stability of phycobiliprotein.

The fourth object of the present invention relates to a phycobiliprotein, which has excellent thermal stability and light stability.

The present invention achieves the above object through the following technical solutions: a phycobiliprotein, including phycobiliprotein, cyanobacteria extract and red algae extract, wherein, the addition ratio of the cyanobacteria extract and red algae extract is the amount of phycobiliprotein 10%-90% of.

The fifth object of the present invention relates to the preparation method of the above-mentioned phycobiliprotein.

The present invention achieves the above object through the following technical solutions: a phycobiliprotein, adding cyanobacteria extract and red algae extract to the phycobiliprotein extract, mixing evenly, and spray drying to obtain the phycobiliprotein. The cyanobacteria extract and the red algae extract are added to the phycobiliprotein extract to maintain the stability of the phycobiliprotein during the high-temperature spray-drying process.

The present invention has the following advantages:

1. The main components of the composition provided by the present invention are cyanobacteria extract and red algae extract, which can greatly improve the thermal stability of phycobiliprotein.

2. The main components of the composition provided by the invention are cyanobacteria extract and red algae extract, which can greatly improve the photostability of phycobiliprotein.

3. Add cyanobacteria extract and red algae extract to the phycobiliprotein extract first, and then spray dry, so as to maintain the stability of phycobiliprotein during the high-temperature spray drying process, so that the phycobiliprotein will not be destroyed and invalidated. Moreover, the addition of cyanobacteria extract and red algae extract will not affect the quality of phycobiliprotein.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Example 1 The preparation method of cyanobacteria extract and the experimental proof of improving the thermal stability of phycobiliprotein

1. Preparation of Spirulina Extract

The preparation method of spirulina extract ①: 1kg of spirulina dry algae powder (or 10kg of spirulina wet algae mud), add 3L deionized water, add 60g/L calcium chloride and 0.2mol/L sodium phosphate, add 10g Cellulase and 10g pectinase were treated at 4°C for 8 hours, sieved to remove algae residue, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain a spiral Algae extract dry powder.

The preparation method of spirulina extract ②: 1kg of spirulina dry algae powder (or 10kg of spirulina wet algae mud), add 3L deionized water, add 2g/L calcium chloride and 300g/L sodium chloride, add 10g fiber Sulfase and 10g pectinase were treated at 4°C for 8 hours, sieved to remove algae residue, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain spirulina Extract dry powder.

Preparation method of spirulina extract ③: 1kg of spirulina dry algae powder (or 10kg of spirulina wet algae mud), add 3L deionized water, add 300g/L sodium chloride and 0.05g/L potassium phosphate, add 10g fiber Sulfase and 10g pectinase were treated at 4°C for 8 hours, sieved to remove algae residue, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain spirulina Extract dry powder.

The preparation method of spirulina extract ④: 1kg spirulina dry algae powder (or 10kg spirulina wet algae mud), add 3L deionized water, add 5g/L sodium chloride, 60g/L calcium chloride and 0.05g/L L sodium phosphate, add 10g cellulase and 10g pectinase, process 8 hours under the condition of 4 degrees, sieve silk filter to remove algae residue, supernatant utilizes the ultrafiltration membrane of aperture 10kDa to filter, collect filtrate, filtrate passes through After spray drying, a dry powder of the spirulina extract is obtained. 2. Preparation of Gexianmi Extract

The preparation method of kudzu rice extract ①: in 1kg dry kudzu rice (or 10kg wet kudzu rice), add 3L deionized water, add 60g/L calcium chloride and 0.2mol/L sodium phosphate salt, add 10g fiber Sulfase and 10g pectinase were treated at 4°C for 8 hours, sieved to remove algae residue, the supernatant was filtered using an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain Gexian Rice extract dry powder.

Preparation method of kudzu rice extract ②: 1kg dry kudzu rice (or 10kg wet kudzu rice), add 3L deionized water, add 2g/L calcium chloride and 300g/L sodium chloride, add 10g cellulose Enzyme and 10g pectinase, treated at 4 degrees for 8 hours, sieved to remove algae residue, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain Ge Xianmi Extract dry powder.

The preparation method of kudzu rice extract ③: in 1kg dry kudzu rice (or 10kg wet kudzu rice), add 3L deionized water, add 300g/L sodium chloride and 0.05g/L potassium phosphate, add 10g cellulose Enzyme and 10g pectinase, treated at 4 degrees for 8 hours, sieved to remove algae residue, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain Ge Xianmi Extract dry powder.

The preparation method of Pueraria kudzu extract ④: In 1kg dry kudzu rice (or 10kg wet kudzu rice), add 3L deionized water, add 5g/L sodium chloride, 60g/L calcium chloride and 0.05g/L Sodium phosphate, add 10g cellulase and 10g pectinase, treat at 4 degrees for 8 hours, sieve to remove algae residue, filter the supernatant with an ultrafiltration membrane with a pore size of 10kDa, collect the filtrate, and spray the filtrate After drying, a dry powder of the kudzu rice extract is obtained.

3. Preparation of Auricularia officinalis extract

Preparation method of ground fungus extract ①: 1kg dry ground fungus (or 10kg wet ground fungus), add 3L deionized water, add 60g/L calcium chloride and 0.2mol/L sodium phosphate, add 10g cellulase and 10g Pectinase, treated at 4 degrees for 8 hours, sieved to remove algae residue, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain the dry powder of the fungus extract .

Preparation method of ground fungus extract ②: 1kg dry ground fungus (or 10kg wet ground fungus), add 3L deionized water, add 2g/L calcium chloride and 300g/L sodium chloride, add 10g cellulase and 10g fruit Gelase, treated at 4°C for 8 hours, sieved to remove algae residue, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain the dry powder of the fungus extract.

Preparation method of ground fungus extract ③: 1kg dry ground fungus (or 10kg wet ground fungus), add 3L deionized water, add 300g/L sodium chloride and 0.05g/L potassium phosphate, add 10g cellulase and 10g fruit Gelase, treated at 4°C for 8 hours, sieved to remove algae residue, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain the dry powder of the fungus extract.

Preparation method of ground fungus extract ④: In 1kg of dry ground fungus (or 10kg of wet ground fungus), add 3L deionized water, add 5g/L sodium chloride, 60g/L calcium chloride and 0.05g/L sodium phosphate, add 10g of cellulase and 10g of pectinase were treated at 4°C for 8 hours, sieved to remove algae residues, the supernatant was filtered with an ultrafiltration membrane with a pore size of 10kDa, the filtrate was collected, and the filtrate was spray-dried to obtain Ground fungus extract dry powder.

Add the cyanobacteria extract to the extract before spray-drying of phycobiliprotein in a ratio of 20%, spray-dry at 180 degrees, collect the spray-dried powder of phycobiliprotein, and use the phycobiliprotein obtained by freeze-drying as a control group, which is called Take 100mg of powder, add 1L of phosphate buffer, and prepare a phycobiliprotein solution with a concentration of 0.1g/L. Measure the maximum absorbance value A ₆₂₀ . The smaller the decrease in A ₆₂₀ , the better the effect of the stabilizer. The results are shown in the table below :

From the results in the table, the maximum absorbance value A ₆₂₀ of the phycobiliprotein obtained by spray drying at 180 degrees remained at about 0.1 in the treatment group without adding cyanobacteria extract, which was nearly 90% lower than the A ₆₂₀ of the control group. After the cyanobacteria extract, the maximum absorbance value A ₆₂₀ is above 0.8, which is close to about 10% compared with the maximum absorbance value A ₆₂₀ of the freeze-dried group, indicating that the thermal stability of phycobiliprotein is greatly improved.

Example 2 The preparation method of red algae extract and the experimental proof of improving the photostability of phycobiliprotein

1. The preparation method of centipede algae extract: 10kg of wet centipede algae is crushed by a grinder, 2L of deionized water is added, the extraction time is heated at 80 degrees for 4 hours, the algae residue is removed by sieve silk, and the supernatant is purified by a pore size of 10kDa. The ultrafiltration membrane is used for filtration, the filtrate is collected, and the filtrate is spray-dried to obtain the dry powder of the centipede algae extract.

2. The preparation method of Dulse palmatum extract: 1kg of dried Dulse palmatum is crushed with a grinder, 2L of deionized water is added, the extraction time is 4 hours at 80 degrees, the algae residue is removed by sieve silk, and the The supernatant liquid is filtered by an ultrafiltration membrane with a pore size of 10 kDa, the filtrate is collected, and the filtrate is spray-dried to obtain a dry powder of the dulse palmate extract.

3. Preparation method of Gracilaria extract: 1 kg of dried Gracilaria is crushed with a grinder, 2L of deionized water is added, the extraction time is heated at 80 degrees for 4 hours, the algae residue is removed by sieve silk, and the supernatant is extracted with a pore size of 10kDa. The ultrafiltration membrane is used for filtration, the filtrate is collected, and the filtrate is spray-dried to obtain dry powder of Gracilaria extract.

Add the red algae extract to the phycobiliprotein extract in a ratio of 20%, and measure the fluorescence decay of the solution with 100 and 200 μmol photons/m ² s light intensity phycobiliprotein for 24 hours. The results are shown in the following table:

From the results in the table, after the red algae extract was added, the relative fluorescence intensity of the phycobiliprotein was above 80% after 24 hours of light, indicating that the photostability of the phycobiliprotein was greatly improved.

Example 3 Photostability Evaluation of a Composition for Enhancing the Photothermal Stability of Phycobiliproteins (hereinafter referred to as Stabilizer)

1. Configure the stabilizer: Spirulina extract: Centipede algae extract = 1:9, add it to the phycobiliprotein extract according to the ratio in the table below, and phycobiliprotein with 100 and 200 μmol photons/m ² s light intensity for 24 hours, Measure the fluorescence decay of the solution, the results are shown in the following table:

From the results in the table, after adding the stabilizer, the relative fluorescence intensity of the phycobiliprotein is above 80% after 24 hours of light, indicating that the photostability of the phycobiliprotein is greatly improved.

2. Configure the stabilizer: Pueraria kudzu extract: Dulse palmiform = 1:9, add it to the phycobiliprotein solution according to the ratio in the table below, and phycobiliprotein with 100 and 200 μmol photons/m ² s light intensity for 24 hours , measure the fluorescence decay of the solution, the results are shown in the following table:

From the results in the table, after adding the stabilizer, the relative fluorescence intensity of the phycobiliprotein is above 80% after 24 hours of light, indicating that the photostability of the phycobiliprotein has been greatly improved.

3. Configure the stabilizer: Auricularia auricula extract: Gracilaria extract = 1:9, add it to the phycobiliprotein solution according to the ratio in the table below, measure the phycobiliprotein with 100 and 200 μmol photons/m ² s light intensity for 24 hours The fluorescence decay of the solution, the results are shown in the following table:

From the results in the table, after adding the stabilizer, the relative fluorescence intensity of the phycobiliprotein is above 80% after 24 hours of light, indicating that the photostability of the phycobiliprotein has been greatly improved.

Example 4 Thermal stability evaluation of a composition that enhances the photothermal stability of phycobiliprotein (hereinafter referred to as stabilizer)

1. Configure stabilizer: Spirulina extract: Centipede algae extract = 9:1, add the phycobiliprotein to the extract before spray-drying of phycobiliprotein according to the ratio in the table below, spray-dry at 180 degrees, and collect different stabilizer compositions The phycobiliprotein powder obtained by freeze-drying was used as the control group. Weighed 100mg powder and added 1L phosphate buffer to prepare a phycobiliprotein solution with a concentration of 0.1g/L. Measure the maximum absorbance value A ₆₂₀ , A ₆₂₀ The smaller the decrease, the better the effect of the stabilizer

2. Configure stabilizer: kudzu extract: dulse palmiform = 1:9, add it to the extract of phycobiliprotein before spray drying according to the ratio in the table, spray dry at 180 degrees, and collect different stabilizers For the powder of the composition, the phycobiliprotein obtained by freeze-drying was used as the control group, 100 mg of the powder was weighed, and 1 L of phosphate buffer was added to prepare a phycobiliprotein solution with a concentration of 0.1 g/L, and the maximum absorbance value A ₆₂₀ was measured. The smaller the decrease of A ₆₂₀ , the better the effect of the stabilizer.

3. Configure the stabilizer: Auricularia auricularia extract: Gracilaria extract = 1:9, add the phycobiliprotein to the extract before spray-drying according to the ratio in the table, spray-dry at 180 degrees, and collect different stabilizer compositions The phycobiliprotein powder obtained by freeze-drying was used as the control group. Weighed 100mg powder and added 1L phosphate buffer to prepare a phycobiliprotein solution with a concentration of 0.1g/L. Measure the maximum absorbance value A ₆₂₀ , A ₆₂₀ The smaller the decrease, the better the effect of the stabilizer. The results are shown in the table below:

As can be seen from the results in the table, after adding the stabilizer, the maximum absorbance value A ₆₂₀ of the phycobiliprotein obtained by spray drying at 180 degrees decreases slightly, indicating that the thermal stability of the phycobiliprotein is greatly improved.

Claims (10)

The composition for enhancing the photothermal stability of phycobiliprotein is characterized by comprising extracts of blue algae and red algae. The composition for enhancing the photothermal stability of phycobiliprotein according to claim 1, characterized in that, the respective mass fractions of the cyanobacteria extract and the red algae extract are: 10%-90% of the cyanobacteria extract, 10%-90% of the red algae extract, Extract 10%-90%. The composition for enhancing the photothermal stability of phycobiliproteins according to claim 1, wherein the cyanobacteria extract is an extract of cyanobacteria biomass, and the cyanobacteria biomass is spirulina, kudzu radices or ground cyanobacteria The fungus is dry algae or wet algae; the red algae extract is the extract of red algae biomass, and the red algae biomass is centipede algae, dulse palmate and Gracilaria, which is dry algae or wet algae. The preparation method of the composition for enhancing the photothermal stability of phycobiliprotein is characterized in that it comprises the following steps: (1) After the cyanobacteria biomass is subjected to hyperosmotic coupling enzymolysis treatment, it is filtered, and the obtained filtrate is an extract, which is purified and dried to obtain a cyanobacteria extract powder; (2) get red algae biomass and adopt hot water extraction method to extract, filter, gained filtrate is extract, purify, dry to obtain red algae extract powder; (3) Take blue algae extract powder and red algae extract powder and mix to obtain. The preparation method of the composition for enhancing the photothermal stability of phycobiliprotein according to claim 4, characterized in that, the hyperosmotic condition of the hypertonic coupling enzymolysis of the step (1) is 5-300g/L chlorination A combination of any two or three of sodium, 2-60g/L calcium chloride and 0.05-0.2mol/L potassium phosphate (sodium) salt, the enzymolysis treatment condition is a compound enzyme system of cellulase and pectinase, Hyperosmotic coupled enzymatic hydrolysis treatment at 4 degrees for 7-9 hours. The preparation method of the composition for enhancing the photothermal stability of phycobiliprotein according to claim 4, characterized in that, the purification in the step (1) and the step (2) is ultrafiltration purification, that is, using a pore size of 10kDa to filter The membrane is subjected to ultrafiltration. The preparation method of the composition that enhances the photothermal stability of phycobiliprotein according to claim 4, is characterized in that, in described step (1) and step (2), drying is that filtrate is subjected to spray drying or low-temperature freeze-drying treatment ; In the step (2), the red algae biomass is first broken into smaller fragments to facilitate subsequent hot water extraction; in the step (2), the hot water extraction is heated at 70-90 degrees for 3-5 hours. Claims 1-3 Use of the composition for enhancing the photothermal stability of phycobiliprotein as an additive for improving the thermal stability and light stability of phycobiliprotein. A phycobiliprotein, including phycobiliprotein, is characterized in that it also includes cyanobacteria extract and red algae extract, wherein the addition ratio of the cyanobacteria extract and red algae extract is 10%-90% of the phycobiliprotein amount %. A preparation method of phycobiliprotein according to claim 9, characterized in that cyanobacteria extract and red algae extract are added to the phycobiliprotein extract, mixed evenly, and then spray-dried to obtain phycobiliprotein.