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WO2023115326A1 - Composition pour améliorer la stabilité photothermique de phycobiliprotéines, son procédé de préparation et son utilisation - Google Patents

Composition pour améliorer la stabilité photothermique de phycobiliprotéines, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2023115326A1
WO2023115326A1 PCT/CN2021/140061 CN2021140061W WO2023115326A1 WO 2023115326 A1 WO2023115326 A1 WO 2023115326A1 CN 2021140061 W CN2021140061 W CN 2021140061W WO 2023115326 A1 WO2023115326 A1 WO 2023115326A1
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Prior art keywords
extract
phycobiliprotein
algae
composition
cyanobacteria
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Ceased
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PCT/CN2021/140061
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English (en)
Chinese (zh)
Inventor
向恩宏
吴嘉仪
练桂花
叶永辉
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Foshan Lanqiang Biological Technology Co Ltd
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Foshan Lanqiang Biological Technology Co Ltd
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Priority to PCT/CN2021/140061 priority Critical patent/WO2023115326A1/fr
Publication of WO2023115326A1 publication Critical patent/WO2023115326A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides

Definitions

  • the invention relates to a composition for enhancing the photothermal stability of phycobiliprotein, as well as a preparation method and application of the composition.
  • 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.
  • phycocyanins often exist in the form of trimers ( ⁇ ) 3 or hexamers ( ⁇ ) 6
  • phycoerythrin often exists in the form of hexamer ( ⁇ ) 6 ⁇
  • the molecular weights of ⁇ , ⁇ and ⁇ 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.
  • 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.
  • Citric acid is a small molecule organic acid that is easily soluble in water and has chelation and pH adjustment properties.
  • 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.
  • excessive use of trehalose and citric acid in phycobiliprotein will affect product quality.
  • 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.
  • One of the objects of the present invention is to provide a composition for enhancing the photothermal stability of phycobiliprotein.
  • 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 preparation method of the composition for enhancing the photothermal stability of phycobiliprotein comprises the following steps:
  • 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
  • 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.
  • 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.
  • the red algal biomass is first broken into smaller fragments to facilitate subsequent hot water extraction.
  • 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 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.
  • 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.
  • Example 1 The preparation method of cyanobacteria extract and the experimental proof of improving the thermal stability of phycobiliprotein
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • Stabilizer a Composition for Enhancing the Photothermal Stability of Phycobiliproteins
  • Example 4 Thermal stability evaluation of a composition that enhances the photothermal stability of phycobiliprotein (hereinafter referred to as stabilizer)
  • 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.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
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Abstract

L'invention concerne une composition pour améliorer la stabilité photothermique de phycobiliprotéines, comprenant un extrait d'algues bleues-vertes et un extrait d'algues rouges. La présente invention concerne en outre un procédé de préparation de la composition pour améliorer la stabilité photothermique de phycobiliprotéines. Le procédé comprend les étapes consistant à : mettre à disposition de la biomasse d'algues bleues-vertes, obtenir une solution d'extraction au moyen d'une enzymolyse combinée à une condition hyperosmotique, et purifier et faire sécher la solution d'extraction pour obtenir une poudre d'extrait d'algues bleues-vertes ; mettre à disposition de la biomasse d'algues rouges, obtenir une solution d'extraction au moyen d'une extraction à l'eau chaude, et purifier et faire sécher la solution d'extraction pour obtenir une poudre d'extrait d'algues rouges ; et mélanger la poudre d'extrait d'algues bleues-vertes et la poudre d'extrait d'algues rouges pour obtenir la composition. L'invention concerne en outre l'utilisation de ladite composition en tant qu'additif pour améliorer la thermostabilité et la photostabilité de phycobiliprotéines. Les extraits d'algues bleues-vertes et d'algues rouges peuvent améliorer de manière efficace la photostabilité et la thermostabilité des phycobiliprotéines, de façon à protéger la stabilité des phycobiliprotéines dans un processus de séchage par pulvérisation à haute température, ce qui empêche les phycobiliprotéines d'être détruites et inactivées.
PCT/CN2021/140061 2021-12-21 2021-12-21 Composition pour améliorer la stabilité photothermique de phycobiliprotéines, son procédé de préparation et son utilisation Ceased WO2023115326A1 (fr)

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