Method for preparing oil-soluble phycocyanobilin through combination of protease and lipase as well as obtained product and application of oil-soluble phycocyanobilin
By using a combination of protease and lipase to prepare oil-soluble phycocyanin, the problem of uneven dispersion of phycocyanin in oil-based products was solved, achieving stability and expanding applications.
Patent Information
- Application Number
- CN202511200401.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-26
- Publication Date
- 2025-12-05
AI Technical Summary
Phycocyanin cannot be uniformly dispersed in oil-based foods due to its water solubility, resulting in layering, sedimentation, and uneven coloring, which limits its application in oil-based products.
The polypeptide chain of phycocyanin was hydrolyzed by protease to release the phycocyanin chromophore, and then esterified with fatty alcohol by lipase catalysis to introduce a lipophilic group, thus preparing oil-soluble phycocyanin.
It realizes the transformation of phycocyanin from water-soluble to oil-soluble, improves its stability and dispersibility in oil-based systems, maintains its color and biological activity, and is suitable for oil-based foods, cosmetics and drug carriers.
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to natural pigment modification, in particular to a method for preparing oil-soluble phycocyanin by using protease-lipase combination, and the product and application thereof. BACKGROUND
[0002] Phycocyanin extracted from blue-green algae such as Spirulina has attracted much attention due to its unique and bright sky blue color, which is extremely rare in nature and can meet the market demand for novel and natural color. Phycocyanin is not only an excellent natural colorant, but also a pigment protein rich in various essential amino acids and has been proven to have multiple biological activities such as antioxidant, anti-inflammatory, and immune enhancement, making it a dual-functioning colorant and nutrient, known as "food diamond", and has great application potential in the field of functional food and beverage.
[0003] However, the wide application of phycocyanin is faced with a fundamental technical bottleneck: its inherent physicochemical properties. The structure of phycocyanin is composed of an apoprotein (alpha and beta polypeptide subunits) and a chromophore-phycocyanobilin covalently linked by a sulfide bond. The hydrophilic polypeptide chain wrapped outside gives it good water solubility, but it is precisely this protein shell that becomes the main limitation of its application. First, this water-soluble property limits its application to water-based or hydrophilic food systems, such as beverages, jelly, yogurt, candy, and ice cream. For the vast oil-based food market, such as chocolate, margarine, shortening, baking fat, candy coating, and oil-based salad dressing, water-soluble phycocyanin cannot be uniformly dispersed, resulting in stratification, precipitation, and uneven color, and in fact, it cannot be applied. The water solubility and instability of phycocyanin are mainly due to its external protein polypeptide chain structure. If the connection between the polypeptide chain and the phycocyanobilin chromophore can be cut off by a mild technical means without destroying the structure of the phycocyanobilin chromophore, and an oilophilic group is introduced, it is expected to realize the transformation from water-soluble to oil-soluble and improve its stability, thereby opening up its application in oil-based products. There is no mature method in the prior art to solve this problem. SUMMARY
[0004] The purpose of the present application is to provide a method for preparing oil-soluble phycocyanin by using protease-lipase combination, which successfully converts water-soluble phycocyanin into oil-soluble phycocyanin, solving the technical problem that phycocyanin cannot be applied to oil-based systems. Another purpose of the present application is to provide the oil-soluble phycocyanin prepared by the method and its application.
[0005] Technical solution: The method for preparing oil-soluble phycocyanin by using protease-lipase combination according to the present application comprises the following steps:
[0006] (1) Enzymatic hydrolysis: water-soluble phycocyanin is hydrolyzed in a buffer solution catalyzed by protease, and after inactivation of the protease, the phycocyanin-peptide complex intermediate is obtained by separation and purification;
[0007] (2) Enzymatic esterification: the intermediate obtained in step (1) is dispersed in a non-polar organic solvent, and esterification is carried out with a fatty alcohol with a chain length of C8 to C18 catalyzed by immobilized lipase to obtain an oil-soluble phycocyanin derivative.
[0008] Preferably, the protease in step (1) is at least one of papain, neutral protease, and trypsin.
[0009] Preferably, step (1) specifically includes: hydrolysis of water-soluble phycocyanin in a phosphate buffer solution with pH 6.5-7.5; the hydrolysis temperature is 45-60°C, and the time is 2-8 hours; after enzyme inactivation, the blue precipitate is separated by centrifugation, washed, and dried.
[0010] Further preferably, in step (1), the protease hydrolysis of phycocyanin adopts the following method: phycocyanin is dissolved in 0.05-0.2M phosphate buffer solution (PBS) (pH range 6.5-7.5) at a concentration of 1%-10% (w / v). The protease is added in the phycocyanin buffer solution at a proportion of 1%-5% (w / w) of the dry weight of phycocyanin, and is gently stirred at a speed of 50-150 rpm, and reacted at 45-60°C for 2-8 hours. The protease can be selected from papain (optimum pH 6.0-7.0), neutral protease (derived from Bacillus subtilis, optimum pH 6.5-7.5), or trypsin (optimum pH 7.5-8.5). After the reaction is completed, the reaction solution is quickly heated to 85-95°C and maintained for 10-15 minutes to completely inactivate the protease and terminate the reaction. The inactivated reaction solution is centrifuged at 4000-8000g at 4°C for 15-30 minutes. The supernatant is discarded, and the blue precipitate (i.e. water-insoluble phycocyanin-peptide complex) at the bottom is collected. The precipitate is resuspended and washed by centrifugation 2-3 times with deionized water. The washed precipitate is vacuum freeze-dried to obtain a dark blue intermediate powder.
[0011] Preferably, the protease in step (1) is added at a proportion of 1%-5% (w / w) of the dry weight of phycocyanin.
[0012] Preferably, the immobilized lipase in step (2) is at least one of Candida antarctica lipase B and Rhizopus lipase.
[0013] Preferably, the non-polar organic solvent in step (2) is at least one of n-hexane, isooctane, cyclohexane, and tert-butanol.
[0014] Preferably, the reaction system in step (2) further comprises or Molecular sieve, added in an amount of 3% to 10% of the total mass of the system. Molecular sieve is added in the esterification reaction to absorb the generated water, thereby improving the reaction conversion rate.
[0015] Preferably, the esterification reaction temperature in step (2) is 40 to 55°C, and the time is 12 to 48 hours.
[0016] Further preferably, in step (2), the lipase-catalyzed esterification of phycocyanobilin is carried out as follows: the dried product obtained in the above reaction step 1 is dispersed in n-hexane, isooctane, cyclohexane or tert-butanol, with a concentration of 1% to 5% (w / v). A saturated or unsaturated fatty alcohol with a chain length of C8 to C18, such as octanol, dodecanol (lauryl alcohol), hexadecanol (cetyl alcohol) or oleyl alcohol, is added. The molar ratio of fatty alcohol to phycocyanobilin intermediate is controlled in the range of 5:1 to 20:1. Dry molecular sieve is added in an amount of 3% to 10% of the mass of the system. or Molecular sieve, used to absorb the byproduct water generated in the reaction, to promote the esterification reaction to move in the direction of generating products. Immobilized lipase (Novozym 435, Lipozyme TL IM or RM IM) is added in a proportion of 5% to 15% (w / w) of the dry weight of the phycocyanobilin intermediate, and the reaction is carried out at 40 to 55°C, with a stirring speed of 120 to 200 rpm, for 12 to 48 hours. After the reaction is completed, the immobilized lipase and molecular sieve are separated from the reaction system by simple filtration or low-speed centrifugation (<1000x g). The filtrate is concentrated by rotary evaporation under reduced pressure at a temperature below 50°C to remove the organic solvent. The crude product obtained after the solvent is removed is a dark blue oil. A small amount of n-hexane or ethanol can be used to wash it to remove unreacted fatty alcohol, and then it is concentrated again under reduced pressure to obtain a high-purity oil-soluble phycocyanobilin product.
[0017] The oil-soluble phycocyanobilin prepared by the method described in the present application.
[0018] The oil-soluble phycocyanobilin described in the present application is used in oil-based food, oil-based cosmetics or oily drug carriers.
[0019] Principle of the invention: The present application first uses protease to hydrolyze the polypeptide chain of phycocyanin, releasing a phycocyanobilin chromophore containing a short peptide chain, and then uses lipase to catalyze the esterification of phycocyanobilin with fatty alcohol, connecting lipophilic fatty alcohol groups to the phycocyanobilin molecule, thereby obtaining an oil-soluble phycocyanobilin product, breaking through the application limitations of phycocyanin in oil-based systems.
[0020] Advantages: Compared with the prior art, the present application has the following obvious advantages:
[0021] 1. Functional transformation: successfully converted water-soluble phycocyanin into oil-soluble phycocyanin, the product is significantly oil-soluble (solubility ≥ 50 mg / mL soybean oil), and can be stably dispersed in oil system.
[0022] 2. Mild reaction conditions: the whole process uses enzyme catalysis, the reaction conditions are mild, effectively protects the structure of phycocyanin chromophore from being destroyed, and ensures the color and biological activity of the product.
[0023] 3. Improved product stability: the thermal stability of the modified phycocyanin at 60°C is increased by 2.3 times, and the retention rate is >85% under light (4000 lux) for 8 hours.
[0024] 4. Environmentally friendly and safe process: biological enzymes are used as catalysts, which have high specificity and produce less by-products. The reaction solvent can be recycled and the process is environmentally friendly. DETAILED DESCRIPTION
[0025] The technical solutions of the present application will be further described below in combination with examples.
[0026] Example 1
[0027] (1) Preparation of oil-soluble phycocyanin
[0028] Take 1g phycocyanin (Zhejiang Binmei Biotechnology Co., Ltd.) and dissolve it in 100mL, 0.1M, pH 6.5 phosphate buffer solution, add 50mg papain (Guangxi Nanning Pangbo Biological Engineering Co., Ltd., enzyme activity ≥800U / mg), which is equivalent to 5% w / w enzyme substrate ratio. Under the condition of 55℃, 100rpm, stir for 4 hours. After hydrolysis, heat the mixture in a 90℃ water bath for 15 minutes to inactivate the papain. Centrifuge the inactivated solution at 6000×g at 4℃ for 20 minutes. Carefully discard the supernatant and collect the blue precipitate. Centrifuge the reaction solution and collect the precipitate. Wash the precipitate with 50mL deionized water twice (resuspend-centrifuge). Vacuum dry the precipitate to obtain 0.61g of dark blue intermediate powder.
[0029] Add the above dried intermediate powder to 100mL n-hexane, and then add 5g dodecanol (lauryl alcohol, Shanghai Maikelin Biotechnology Co., Ltd.) and 1g Novozym 435 immobilized lipase (Novozymes, Denmark) and 2.0g dry Molecular sieves, 45°C, 150 rpm shaker for 24 hours. After reaction, filter through a Buchner funnel to recover Novozym 435 and molecular sieves. The filtrate was rotary evaporated at 45°C, -0.08 MPa to remove n-hexane. To the obtained blue oil, 50 mL of anhydrous ethanol was added, stirred to wash off excess lauryl alcohol, and then rotary evaporated again to remove ethanol. Finally, 0.58 g of dark blue oil product was obtained.
[0030] (2) Product application test
[0031] The oil-soluble phycocyanobilin prepared above was added to melted white chocolate (cocoa butter content 35%) at an amount of 0.5% (w / w) and stirred evenly. After cooling and shaping, a blue chocolate with uniform color was obtained. The chocolate was placed at room temperature in the dark for 30 days, and no obvious color change was observed, indicating that it has good application performance and stability.
[0032] Example 2
[0033] (1) Preparation of oil-soluble phycocyanobilin
[0034] 1 g of phycocyanin (Inner Mongolia Tianqi Biotechnology Co., Ltd.) was dissolved in 100 mL of 0.1 M, pH 7.0 phosphate buffer solution to prepare a 3% (w / v) phycocyanin solution. 60 mg of neutral protease (Shanghai Yuanye Biotechnology Co., Ltd.) was added to the solution (neutral protease derived from Bacillus subtilis, enzyme activity ≥200 U / mg), and the amount added was 8,000 U / g of phycocyanin. The solution was stirred at 60°C and 120 rpm for 5 hours. After the reaction was completed, the mixture was heated in a 95°C water bath for 10 minutes to inactivate the enzyme. The mixture was centrifuged at 5000 x g at 4°C for 25 minutes, and the blue precipitate was collected. The precipitate was washed with deionized water three times. After vacuum drying, 0.6 g of dark blue intermediate powder was obtained.
[0035] The dried precipitate was added to 100 mL of isooctane, and then 6 g of oleyl alcohol (National Pharmaceutical Group Chemical Reagent Co., Ltd.), 1.2 g of Lipozyme TL IM immobilized lipase (Novozymes Denmark) and 3.0 g of dry Molecular sieves, 50°C, 180 rpm shaker for 36 hours. After reaction, filter to recover enzymes and molecular sieves. The filtrate was rotary evaporated and washed with n-hexane to obtain 0.81 g of dark blue viscous oil product.
[0036] (2) Product stability test
[0037] The oil-soluble phycocyanin obtained in Example 2 was dissolved in soybean oil to prepare a 0.1 mg / mL solution, and its photostability was compared with that of an unmodified phycocyanin aqueous solution (at the same concentration). After continuous irradiation at 4000 lux for 8 hours, the pigment retention rate of the oil-soluble phycocyanin reached over 85%, while the pigment retention rate of the phycocyanin aqueous solution was less than 40%, indicating a significantly improved photostability.
[0038] Example 3
[0039] (1) Preparation of oil-soluble phycocyanin
[0040] Dissolve 1g of phycocyanin (from Fujian Xiapu Lvqi Algae Industry Co., Ltd.) in 100mL of 0.05M phosphate buffer solution (pH 6.5) to prepare a 10% (w / v) phycocyanin solution. Add 40mg of trypsin (from the USA).
[0041] The enzyme (Sigma-Aldrich, 250 U / g) was stirred at 48°C and 80 rpm for 6 hours. After the reaction, the mixture was heated in a 95°C water bath for 8 minutes to inactivate the enzyme. The solution was centrifuged at 4000×g for 15 minutes at 4°C, and the blue precipitate was collected. The precipitate was collected and washed three times with deionized water. After vacuum drying, 0.65 g of dark blue intermediate powder was obtained.
[0042] The dried precipitate was added to 100 mL of tert-butanol, along with 4 g of octanol (Tianjin Kemeo Chemical Reagent Co., Ltd.), 1 g of Lipozyme RM IM immobilized lipase (Novozymes, Denmark), and 1.5 g of dried precipitate. Molecular sieves were used. The reaction was carried out at 48°C and 200 rpm in a shaker for 48 hours. After the reaction, the enzyme and molecular sieves were recovered by filtration. The filtrate was then subjected to rotary evaporation and washing with n-hexane to obtain 0.33 g of a dark blue viscous oily product.
[0043] (2) Product application testing
[0044] The oil-soluble phycocyanin obtained in Example 3 was added to margarine, giving it a uniform light blue color. After being stored at 4°C for 60 days, the margarine showed no adverse changes in color or flavor, demonstrating its promising application prospects in emulsified fat products.
Claims
1. A method for preparing oil-soluble phycocyanobilin by using a combination of protease and lipase, characterized in that, The method comprises the following steps: (1) enzymatic hydrolysis: water-soluble phycocyanin is hydrolyzed in a buffer solution by a protease, and after inactivation of the protease, the phycocyanin-peptide complex intermediate is separated and purified; (2) enzymatic esterification: the intermediate obtained in step (1) is dispersed in a non-polar organic solvent, and esterification is carried out with a fatty alcohol with a chain length of C8 to C18 using an immobilized lipase as catalyst to obtain an oil-soluble phycocyanin derivative.
2. The method of claim 1, wherein the oil-soluble phycocyanin is prepared by using a combination of a protease and a lipase. The protease in step (1) is at least one of papain, neutral protease, and trypsin.
3. The method of claim 1, wherein the oil-soluble phycocyanobilin is prepared by using a combination of a protease and a lipase. Step (1) specifically comprises: hydrolysis of water-soluble phycocyanin in a phosphate buffer solution at pH 6.5-7.5; the hydrolysis temperature is 45-60℃, and the time is 2-8 hours; after enzyme inactivation, the blue precipitate is separated by centrifugation, washed and dried.
4. The method of claim 1, wherein the oil-soluble phycocyanin is prepared by using a protease-lipase combination. The protease in step (1) is added in an amount of 1%-5% (w / w) of the dry weight of phycocyanin.
5. The method of claim 1, wherein the oil-soluble phycocyanobilin is prepared by using a combination of a protease and a lipase. The immobilized lipase in step (2) is at least one of Candida antarctica lipase B and Rhizopus lipase.
6. The method of claim 1, wherein the oil-soluble phycocyanin is prepared by using a combination of a protease and a lipase. The non-polar organic solvent in step (2) is at least one of n-hexane, isooctane, cyclohexane, and tert-butyl alcohol.
7. The method of claim 1, wherein the oil-soluble phycocyanobilin is prepared by using a combination of a protease and a lipase. The reaction system in step (2) further comprises or Molecular sieve, added in an amount of 3% to 10% of the total mass of the system.
8. The method of claim 1, wherein the oil-soluble phycocyanobilin is prepared by using a combination of a protease and a lipase. The esterification reaction temperature in step (2) is 40-55℃, and the time is 12-48 hours.
9. An oil-soluble phycocyanin prepared by the method of any one of claims 1-9.
10. Use of the oil-soluble phycocyanin of claim 9 in oil-based food, oil-based cosmetics, or oily drug carriers.